# Domestic use of pesticides during early periods of development and risk of testicular germ cell tumors in adulthood: a French nationwide case-control study

Aurélie Danjou, Olivia Pérol, Astrid Coste, Elodie Faure, Rémi Béranger, Helen Boyle, Elodie Belladame, Lény Grassot, Matthieu Dubuis, Johan Spinosi, et al.

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Aurélie Danjou, Olivia Pérol, Astrid Coste, Elodie Faure, Rémi Béranger, et al. Domestic use of pesticides during early periods of development and risk of testicular germ cell tumors in adulthood: a French nationwide case-control study. Environmental Health, 2021, 20 (111), 10.1186/s12940-021-00795-y. hal-03421589

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# Domestic use of pesticides during early periods of development and risk of testicular germ cell tumors in adulthood: a French nationwide case-control study

Aurélie M. N. Danjou¹* , Olivia Pérol²,³, Astrid Coste²,³, Elodie Faure²,⁴, Rémi Béranger⁵, Helen Boyle⁶, Elodie Belladame², Lény Grassot², Matthieu Dubuis², Johan Spinosi⁷, Liacine Bouaoun¹, Aude Fléchon², Louis Bujan⁸,⁹, Véronique Drouineaud⁹,¹⁰, Florence Eustache⁹,¹¹, Isabelle Berthaut⁹,¹¹,¹², Jeanne Perrin⁹,¹³,¹⁴, Florence Brugnon⁹,¹⁵,¹⁶, Barbara Charbotel²,¹⁷, Joachim Schüz¹ and Béatrice Fervers²,³For the TESTIS study group

**Abstract**

**Background:** Testicular germ cell tumours (TGCT) are the most frequent cancers in young men in developed countries and their incidence rate has doubled worldwide over the past 40 years. Early life exposures to pesticides are suspected to increase TGCT risk. Our research aimed at estimating adult TGCT risk associated with parental domestic use of pesticides during early periods of child development.

**Methods:** We conducted a case-control study of 304 TGCT cases, aged 18–45 years old, recruited in 20 French university hospitals, and 274 controls frequency-matched on hospital and birth year. Participants’ mothers provided information on their domestic use of pesticides from 1 year before start of pregnancy to 1 year after their son’s birth, for gardening activities, treatment of indoor plants, pets, wood and mold, and pest control. Odds ratios (OR) for TGCT (overall and by histological subtype) and 95% confidence intervals (CI) were estimated using conditional logistic regression.

**Results:** Prevalence of reported domestic use of pesticides was 77.3% for insecticides, 15.9% for fungicides and 12.1% for herbicides. While no association was found for any use of insecticides (OR = 1.27, CI = 0.80–2.01) or herbicides (OR = 1.15, CI = 0.67–2.00), elevated risks of TGCT overall (OR = 1.73, CI = 1.04–2.87) and non-seminoma subtype (OR = 2.44, CI = 1.26–4.74) were observed for any use of fungicides. When specific purposes were examined, using fungicides and/or insecticides for woodwork (OR = 2.35, CI = 1.06–5.20) and using insecticides on cats and dogs (OR = 1.95, CI = 1.12–3.40) were associated with increased risk of non-seminoma subtype. We found no association for seminoma subtype.

**Conclusions:** Although recall bias may partially explain the elevated ORs, our study provides some evidence of a positive association between domestic use of pesticides during early periods of development, particularly fungicides

<footnote>*Correspondence: DanjouA@fellows.iarc.fr
¹Environment and Lifestyle Epidemiology Branch, International Agency for Research on Cancer/World Health Organization, 150 cours Albert Thomas, 69372 Lyon, Cedex 08, France
Full list of author information is available at the end of the article</footnote>
and risk of adult TGCT and non-seminoma. Given the common domestic use of pesticides in France, further research on TGCT risk is warranted.

**Keywords:** Case-control study, Domestic use, Epidemiology, Environment, Pesticides, Testicular germ cell tumor

## Background

While overall testicular germ-cell tumours (TGCT) are a rare cancer, accounting for 1% of all neoplasms, these are the most common cancer occurring in young men aged 15 to 45 years in developed countries, in particular among Caucasian populations. Their incidence rate has importantly increased over the past 40 years [1–3]. TGCT in young men represent 94–98% of testicular cancers [1, 4]. These are preceded by germ cell neoplasia in situ (GCNIS), and comprise two main histological subtypes, seminomas (SE) and non-seminomas (NS), which also include mixed tumours.

Only few risk factors for TGCT have been established to date – i.e. genetic predisposition and previous history of testicular cancer – and alone they do not explain the increase in TGCT incidence rates over time, nor the differences observed between population groups and geographic regions [5, 6]. Pre- and perinatal factors are putative risk factors because GCNIS-related TGCT occur relatively early in life and have been found associated with other male reproductive disorders starting during foetal life, including congenital malformations (cryptorchidism and hypospadias), which could be signs of a common testicular dysgenesis syndrome (TDS) [7]. Environmental exposures to pesticides with endocrine disrupting properties have been suggested to be determinants of TGCT [8, 9].

Pesticides are defined as “any substance or mixture of substances of chemicals or biological ingredients intended for repelling, destroying or controlling any pest, or regulating plant growth” [10]. Multiple sources contribute to the overall pesticide exposure, including occupation, residence in the vicinity of agricultural areas, contaminated food intake and drinking water, and domestic use (indoor, in the garden or on pets). Domestic use of pesticides appears common and a non-negligible source of exposure in the general population. In 2014, 75% of the French households interviewed reported having used pesticide products at home at least once during the preceding year [11]. In a study on pesticide hair contamination in pregnant women, approximately one third of the most concentrated pesticides detected were pesticides with domestic usage [12]. So far, very few studies have examined the association between domestic use of pesticides and risk of TGCT. The use of pesticides during gardening activities and household use of insecticides have been found associated with increased risks of TGCT

and NS in studies investigating exposures during adulthood [13–15]. To our knowledge, no study has focused on early periods of development, namely preconception, pregnancy and early infancy, whereas the first trimester of pregnancy has been identified as the masculinization programming window [16–19]. Domestic use of pesticides during pregnancy or around birth has been found to be associated with other cancers in the offspring, especially childhood leukemia and brain tumors [20, 21].

Here we report results from a French nationwide case-control study designed to investigate whether early life exposures to environmental risk factors and in particular pesticide use may increase the risk of TGCT in young men. This research aimed at estimating the risk of TGCT in adulthood associated with domestic use of pesticides during early periods of development, covering the period ranging from 1 year before start of pregnancy to 1 year after birth. The research further investigated the association according to TGCT histological subtypes.

## Methods

### Study design

A multicenter prospective case-control study was conducted between January 2015 and April 2018 in 20 out of the 23 university hospital centers in Metropolitan France. The study protocol has been described previously [22]. Briefly, the study included patients diagnosed with primary GCNIS-related TGCT, aged 18 to 45 years, and referred for semen preservation prior to TGCT treatment to the regional sperm banks located in the university hospitals affiliated with the French national network of study and preservation centers for eggs and semen (Fédération Française des Centres d'étude et de conservation des oeufs et du sperme, CECOS). Recruitment of TGCT cases had to have been performed within 12 months of diagnosis. Two groups of controls, with no personal history of testicular cancer or cryptorchidism as it may be associated with higher risk of TGCT occurrence, were recruited and frequency-matched to cases on year of birth (+/- 3 years) and hospital center. Group A controls were sperm donors and partners of women consulting for fertility disorders, with normal sperm production (total count ≥39M sperm cells per ejaculate) and recruited in CECOS and in assisted reproduction treatment (ART) centers respectively. Group B controls were partners of women treated for a pathological pregnancy in specialized maternity clinics equipped with maternal
and neonatal intensive care units, adjacent to hospital centers. Referral and recruitment of cases and both groups of controls were regional. Participants born in Metropolitan France were eligible.

After written consent, participants received a hand-out to prepare for the interview. In addition, they were asked to provide written permission to contact their biological mother or the closest relative in case the mother was not available. Trained investigators (IPSOS Company) conducted a 90-min telephone interview with the participants, blinded to the case-control status, and using a structured, pretested and computer assisted questionnaire [23]. Upon agreement of the participant, the mother/relative was invited to participate in the study by telephone interview, using the same procedures as for the participants.

Data collected from participants included residential history from birth onwards, lifetime occupational history, including workplace addresses and information on specific exposures for each job (pesticides, solvents, metals and welding fumes, and plastic), socio-economic status, birth characteristics, medical history and lifestyle factors (smoking status and drug use). Participants' mothers/relatives who consented to participate provided similar information as well as additional data covering pregnancy and postnatal periods (treatments, age, morphology, breastfeeding), and reported the occupational history of the father from 1 year before conception to when the son turned 17 years old. Moreover, the questionnaire comprised items related to domestic use of pesticides, by participants at puberty and young adulthood, and by mothers from 1 year before start of pregnancy to 1 year after the son's birth and at puberty. Sixty percent (N=698) of the participants provided two blood specimens at inclusion that have been frozen and stored for latter analysis.

Both participants and mothers/relatives provided written informed consent prior to entry in the study. Participants were compensated for answering the questionnaire (20€ in gift voucher) and providing blood samples (additional 20€). The study received ethical approval from the French Ethics Committee (ref. no. A14-94), the French national agency for medicines and health products safety (ref. no. 140184B-12) and the IARC Ethics Committee (ref. no. 14-26), and was declared to the *Commission nationale Informatique et Libertés* (MR-001, ref. no. 2016-177).

## Ascertainment of GCNIS-related TGCT cases

TGCT cases were histologically confirmed by review of pathology reports and serum tumor markers (84%) by a TGCT expert (HB), and classified as seminomas or non-seminomas (embryonal carcinoma, choriocarcinoma,

yolk sac tumor, teratoma and mixed germ cell tumor) according to the International Classification of Disease for Oncology (ICD-O) and the WHO classification of tumors of the urinary system and male genital organs [4, 24]. Patients with testicular cancer not originating from GCNIS were excluded (spermatocytic tumors, epidermoid cysts, neuroendocrine tumors, Leydig cell tumors, Sertoli cell tumors and hemangiomas). Because the proportion of false-positive TGCT was low (5.1%), TGCT not confirmed due to missing pathology report (7.8%, N=43) were included as cases in our analyses.

## Study population

Overall, 1463 eligible subjects have been invited to participate in the study, among which 1367 (93.4%) agreed to participate: 550 TGCT cases, 447 group A and 370 group B controls. Among the total 96 subjects that refused to participate, the most frequent reasons for non-participation were lack of interest in the study (n=32; 33%), no wish to participate (n=27; 28%), or lack of availability (n=20; 21%) (Figure S1). The proportion of men aged 25 years or younger was higher in the non-participants than in the participants (29% versus 11%), while the proportion of men aged 31–35 years old was higher in the participants than in the non-participants (33% versus 21%). In the non-participating men, the proportion of employment in intermediate occupations was higher and the proportion of “not professionally active” was lower than among the eligible subjects that agreed to participate in the study (23% versus 11, and 18% versus 56%, respectively, data not shown). Among the 1367 enrolled men, 853 (62%) agreed to contact their mothers and finally 640 mothers/relatives agreed to participate. Of the subjects that agreed to participate, 44 participants were excluded for not meeting inclusion criteria: N=25 cases were not confirmed by pathology reports (N=21 non GCNIS-related TGCT; N=4 with absence of a tumor); N=5 confirmed GCNIS-related TGCT with time from diagnosis to study inclusion >12 months (N=4) or with missing date of diagnosis (N=1); N=1 group B control not born in Metropolitan France; and N=13 controls (8 group A and 5 group B) who reported personal history of cryptorchidism. Among eligible subjects (N=1323), 168 did not complete the telephone interview and were excluded (N=48 TGCT cases, N=46 group A controls and N=74 group B controls); reasons for this included refusal (N=44), unreachable subjects after three telephone calls (N=123) and 1 person who passed away prior to interview (Figure S1).

A total of 1124 participants completed the interview, as well as 31 participants' mothers for whom their son was not interviewed (N=1155). The study population was composed of participants for whom mothers/relatives
had completed the interview, and finally included 570 participants' mothers and 8 participants' relatives (N=578, 50% participation): 304 TGCT cases – 144 SE (47%) and 132 NS (43%)–, 145 group A controls and 129 group B controls.

## Exposure assessment

Domestic use of pesticides at any time from 1 year before start of pregnancy to 1 year after the son's birth was collected during the interview with participants' mothers/relatives through 22 structured questions. The use of insecticides, herbicides and fungicides was assessed for gardening activities, treatment of indoor plants, pest control (against flying insects, crawling insects or termites), treatment of pets, for woodwork and against molds, as well as practice of each activity. Interviewers could provide definition for all items upon request of the interviewee. Frequency of pesticide use was further recorded for pest control (never; less than once a month; less than once a week; more than once a week; daily). Direct use (by the mother/relative) or indirect use (by another person sharing the household, the mother's spouse for instance) of pesticides was specified for gardening activities and treatment of indoor plants. Furthermore, we computed dichotomous exposure variables related to indoor use of pesticides (ever/never) which comprised treatment of indoor plants, pest controls, treatment of pets, woodwork and molds, and outdoor uses of pesticides (ever/never) that were based on gardening activities and split according to direct or indirect use.

## Statistical analysis

Participants' characteristics were described by case-control status using mean and SD for continuous variables and frequency and percentage for categorical variables. Odds ratios (OR) and corresponding 95% confidence intervals (CIs) for TGCT in adulthood were estimated for domestic use of pesticides during early periods of development (never use as reference category), using conditional logistic regression models [25]. All models were conditioned for matching factors (hospital center and birth year grouped in 5-year categories) [26]. Trend tests were performed for the ordinal variables reporting frequency of pesticides use. Covariates suspected to be early life risk factors for TGCT were first identified from the literature [27] and considered for adjustment: maternal exposure to diethylstilbestrol during pregnancy (yes, no); maternal smoking during pregnancy (yes, no); breastfeeding (yes, no); personal history of inguinal hernia (yes, no); birth weight (<2500, 2500–3999, ≥3999 g); gestational age (≤36, >36 weeks of pregnancy); birth order (first, second, third, fourth and more); sibship size (one, two, three, four and more brothers and sisters);

multiple birth (yes, no); family history of testicular cancer (yes, no); family history of cryptorchidism (yes, no); geographic origin (French by birth, by acquisition). In addition, parental occupations at birth and presence of crops in the vicinity of the birthplace were assessed. Parental jobs and industries were coded by a hygienist according to ISCO-1968 (International Standard Classification of Occupations) and NAF-1999 (Nomenclature d'activités française). Presence of crops – i.e. arable lands, vineyards, and orchards – in a 500 m buffer around the birthplace (yes, no) was assessed using a geographic information system (GIS) and automatic processing of historical aerial images integrated into the Gouramic software [28]. Each previous suspected covariate was then sequentially added to the model including the exposure variable, and change in OR comparing models with and without the additional covariate was then computed; covariates were retained if the change in OR was >10%. As none of the covariates assessed led to a change in OR >10% (Table S1), results presented were computed from models not adjusted for those covariates. As matching on birth year grouped in 5-year intervals resulted in large strata, additional adjustments were performed on age (at diagnosis for cases and inclusion for controls) as a continuous variable to avoid residual confounding by age within the birth year groups.

We estimated OR for TGCT and 95% confidence intervals (CI) according to the histological subtype of the tumors (seminoma and non-seminoma) and tested the heterogeneity of associations using polytomous logistic regression for matched case-control studies (SAS macro %subtype) [29]. P-values for heterogeneity were derived from the likelihood ratio test [29]. Histological subtype was missing for N=27 cases.

We conducted stratified analysis according to birth cohorts (1970–1980, 1981–1990, and 1991–1999) due to changes in pesticide usage and sales over time. Models stratified by birth cohorts were not conditioned for birth year to avoid over-fitting. Because women living in rural areas, i.e. in the vicinity of agricultural activities, might be exposed to pesticides at higher levels than women from urban areas [30], models were stratified according to the urban or rural status of the birthplace. The urban/rural status of the birthplace was determined according to the population size of the town: birthplaces with less than 2000 inhabitants were considered as rural, whereas the others were urban [31]. Analyses were further stratified on the season during which first trimester of pregnancy occurred (spring/summer, autumn/winter), as prevalence of pesticide use is higher during spring and summer seasons [19, 32]. Finally, the association with the use of pesticides in gardening by the mother/relative and/or another person from the household was stratified by the reported gardening activities of the mother/relative
(yes, no) to consider potential exposure due to re-entry
tasks during gardening [33, 34]. Effect modifications
between domestic use of pesticides and each of the above
mentioned strata variables were tested with likelihood
ratio tests comparing models with and without interac-
tion terms [25].

The following sensitivity analyses were performed. First, we investigated the effect of domestic use of pesticides on TGCT excluding cases with personal history of cryptorchidism (N=19), and in a second time cases and controls who reported family history of cryptorchidism (N=26) and testicular cancer (N=36). Third, we excluded the TGCT cases not confirmed by pathology reports (N=27) and models were run in this subgroup. We further excluded the participants for which a close relative and not the mother (n=8) answered the interview questions in order to minimize misclassification of exposures during pregnancy.

Domestic exposures to pesticides were only reported if there were at least five exposed cases or controls in exposure categories. For cell counts of five or less, we aggregated cell counts across categories when possible. If the number of exposed cases or controls was less than five and categories could not be combined, risk estimates were not reported because of statistical uncertainty of estimates.

All *P*-values were two-sided and the significance level was set at 0.05. SAS statistical software version 9.4 (SAS Institute Inc., Cary, NC, USA) was used for data analysis.

Results

The main characteristics of TGCT cases and group A and
group B controls are shown in Table 1. Average mater-
nal age at birth was 27.6, 27.0 and 27.8 years, respectively.
Low birthweight (< 2500 g) was reported for 6.3% of cases
and 5.5%/4.4% of group A and group B controls respec-
tively. The majority of participants were first born (40.5
to 48.1%), and few cases and controls were born from a
multiple pregnancy (1.4 to 3.3%). There was no difference
between TGCT cases and controls in prenatal and peri-
natal factors, in parental occupations and in birthplace
characteristics. For predisposing factors, family history
of testicular cancer was more frequent among TGCT
cases than controls (8.2% versus 3.4%/4.7%). There was
no difference between the two groups of A and B con-
trols, except for paternal job at birth. Age distribution at
diagnosis/ inclusion was slightly different between cases
and controls, as there were more cases than controls aged
less than 25 years old and more controls than cases aged
31 to 35 years old (Table 2). Among TGCT cases, 43.4%
were NS and 47.4% were SE. Age at diagnosis peaked at
26–30 years old for NS and 31–35 years old for SE.

A comparison of subjects' characteristics of mothers/
relatives interviewed (N=578), and of mothers not inter-
viewed, did not show differences in terms of participants'
pre- and perinatal factors or socio-economic status at
interview, nor differences of mothers' socio-economic
status at subjects' birth. Family history of TGCT was
more frequent in sons of mothers interviewed. As the
participation rate differed between case mothers (52.6%)
and control mothers (40.1%), there was a higher propor-
tion of cases in the group of mothers interviewed and age
distribution of cases and controls was different between
the two groups (Table S2). Prevalence of domestic use of
pesticides from 1 year before start of pregnancy to 1 year
after birth in the study population (N=578) was 77.3%
for insecticides, 15.9% for fungicides, 12.1% for herbi-
cides and 78.9% for pesticides overall (Table 3, Table S3).
As group A and group B controls were not significantly
different based on socio-demographic, pre-, perinatal and
predisposing characteristics, there were grouped in the
analyses. There was no association between TGCT risk
and domestic use of insecticides (OR=1.27, CI=0.80–
2.01), herbicides (OR=1.15, CI=0.67–2.00), as well as
pesticides overall (OR=1.18, CI=0.74–1.88) (Table 3,
Table S3). We observed statistically significant increased
TGCT risk (OR=1.73, CI=1.04–2.87) and NS risk
(OR=2.44, CI=1.26–4.74) for the domestic use of fun-
gicides, while no association was found for SE. Adjust-
ment on age for main exposures and outcomes showed
slightly lower associations as observed in the main analy-
sis (Table S4). The observed association between domes-
tic use of fungicides and TGCT risk provided similar
OR but was no longer statistically significant (OR=1.55,
CI=0.91–2.65). However, the association observed
for domestic use of fungicides and risk of NS remained
unchanged (OR=2.40, CI=1.17–4.91) (Table S4).

Tables 4 and S5 present the ORs for domestic use of
pesticides according to specific household applications,
for exposure categories with at least 5 cases and 5 con-
trols. No association was found for the domestic use of
pesticides during gardening activities, for treatment of
indoor plants, and for pest control. We observed a sta-
tistically significant increased OR for NS associated with
the use of fungicides and/or insecticides for woodwork
(OR=2.35, CI=1.06–5.20) with heterogeneity in this
association for NS versus SE (p-for-heterogeneity=0.03).
The use of insecticides on cats and dogs was associ-
ated with a statistically significant increase in NS risk
(OR=1.95, CI=1.12–3.40) and was found different from
the association with SE (p-for-heterogeneity=0.01). No
significant trends were noticed with insecticides use fre-
quency against crawling or flying insects respectively
(Table 4). Indoor use and direct outdoor use of pesti-
cides were not associated with TGCT risk (OR=1.14,
<table><caption>**Table 1** Characteristics of TGCT cases and controls (group A and group B), N = 578, case-control study, France, 2015–2018</caption><thead><tr><th></th><th>TGCT cases (N = 304)</th><th>Group A controls (N = 145)</th><th>Group B controls (N = 129)</th></tr><tr><th></th><th>n (%)</th><th>n (%)</th><th>n (%)</th></tr></thead><tbody><tr><td colspan="4"><strong>Prenatal Characteristics</strong></td></tr><tr><td><strong>Maternal use of diethylstilbestrol during pregnancy</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>296 (97.4)</td><td>142 (97.9)</td><td>129 (100.0)</td></tr><tr><td>Yes</td><td>1 (0.3)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr><tr><td>Missing</td><td>7 (2.3)</td><td>3 (2.1)</td><td>0 (0.0)</td></tr><tr><td><strong>Maternal smoking during pregnancy</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>252 (82.9)</td><td>122 (84.1)</td><td>110 (85.3)</td></tr><tr><td>Yes</td><td>52 (17.1)</td><td>23 (15.9)</td><td>19 (14.7)</td></tr><tr><td><strong>Maternal age (years), mean ± SD</strong></td><td>27.6 ± 4.7</td><td>27.0 ± 4.7</td><td>27.8 ± 4.6</td></tr><tr><td><strong>Paternal age (years), mean ± SD</strong></td><td>29.8 ± 4.8</td><td>29.5 ± 5.3</td><td>30.2 ± 5.4</td></tr><tr><td>Missing</td><td>169 (55.6)</td><td>80 (55.2)</td><td>62 (48.1)</td></tr><tr><td colspan="4"><strong>Perinatal Characteristics</strong></td></tr><tr><td><strong>Inguinal hernia</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>263 (86.5)</td><td>128 (88.3)</td><td>117 (90.7)</td></tr><tr><td>Yes</td><td>23 (7.6)</td><td>8 (5.5)</td><td>7 (5.4)</td></tr><tr><td>Missing</td><td>18 (5.9)</td><td>9 (6.2)</td><td>5 (3.9)</td></tr><tr><td><strong>Birth weight</strong></td><td></td><td></td><td></td></tr><tr><td>&lt;2500 g</td><td>19 (6.3)</td><td>8 (5.5)</td><td>6 (4.7)</td></tr><tr><td>[2500–4000 g]</td><td>250 (82.2)</td><td>122 (84.1)</td><td>106 (82.2)</td></tr><tr><td>&gt;=4000 g</td><td>30 (9.9)</td><td>15 (10.3)</td><td>16 (12.4)</td></tr><tr><td>Missing</td><td>5 (1.6)</td><td>0 (0.0)</td><td>1 (0.8)</td></tr><tr><td><strong>Gestational age</strong></td><td></td><td></td><td></td></tr><tr><td>&lt;=36 weeks</td><td>27 (8.9)</td><td>10 (6.9)</td><td>8 (6.2)</td></tr><tr><td>&gt; 36 weeks</td><td>270 (88.8)</td><td>134 (92.4)</td><td>120 (93.0)</td></tr><tr><td>Missing</td><td>7 (2.3)</td><td>1 (0.7)</td><td>1 (0.8)</td></tr><tr><td><strong>Birth order</strong></td><td></td><td></td><td></td></tr><tr><td>First</td><td>123 (40.5)</td><td>61 (42.1)</td><td>62 (48.1)</td></tr><tr><td>Second</td><td>101 (33.2)</td><td>49 (33.8)</td><td>41 (31.8)</td></tr><tr><td>Third</td><td>49 (16.1)</td><td>23 (15.9)</td><td>15 (11.6)</td></tr><tr><td>Fourth and more</td><td>31 (10.2)</td><td>12 (8.3)</td><td>11 (8.5)</td></tr><tr><td><strong>Sibship size</strong></td><td></td><td></td><td></td></tr><tr><td>1</td><td>21 (6.9)</td><td>23 (15.9)</td><td>14 (10.9)</td></tr><tr><td>2</td><td>148 (48.7)</td><td>48 (33.1)</td><td>55 (42.6)</td></tr><tr><td>3</td><td>96 (31.6)</td><td>48 (33.1)</td><td>40 (31.0)</td></tr><tr><td>&gt;=4</td><td>39 (12.8)</td><td>26 (17.9)</td><td>20 (15.5)</td></tr><tr><td><strong>Birth from multiple pregnancy</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>276 (90.8)</td><td>134 (92.4)</td><td>122 (94.6)</td></tr><tr><td>Yes</td><td>10 (3.3)</td><td>2 (1.4)</td><td>3 (2.3)</td></tr><tr><td>Missing</td><td>18 (5.9)</td><td>9 (6.2)</td><td>4 (3.1)</td></tr><tr><td><strong>Birth cohort</strong></td><td></td><td></td><td></td></tr><tr><td>1970–1980</td><td>89 (29.3)</td><td>47 (32.4)</td><td>34 (26.4)</td></tr><tr><td>1981–1990</td><td>162 (53.3)</td><td>88 (60.7)</td><td>88 (68.2)</td></tr><tr><td>1991–2000</td><td>53 (17.4)</td><td>10 (6.9)</td><td>7 (5.4)</td></tr><tr><td colspan="4"><strong>Predisposing Characteristics</strong></td></tr><tr><td><strong>Geographic origin</strong></td><td></td><td></td><td></td></tr><tr><td>French by birth</td><td>284 (93.4)</td><td>136 (93.8)</td><td>123 (95.3)</td></tr><tr><td>French by acquisition</td><td>2 (0.7)</td><td>0 (0.0)</td><td>2 (1.6)</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th></th><th>TGCT cases (N = 304)</th><th>Group A controls (N = 145)</th><th>Group B controls (N = 129)</th></tr><tr><th></th><th>n (%)</th><th>n (%)</th><th>n (%)</th></tr></thead><tbody><tr><td>Missing</td><td>18 (5.9)</td><td>9 (6.2)</td><td>4 (3.1)</td></tr><tr><td><strong>Family history of TGCT</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>260 (85.5)</td><td>131 (90.3)</td><td>119 (92.2)</td></tr><tr><td>Yes</td><td>25 (8.2)</td><td>5 (3.4)</td><td>6 (4.7)</td></tr><tr><td>Missing</td><td>19 (6.3)</td><td>9 (6.2)</td><td>4 (3.1)</td></tr><tr><td><strong>Family history of cryptorchidism</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>264 (86.8)</td><td>131 (90.3)</td><td>122 (94.6)</td></tr><tr><td>Yes</td><td>19 (6.3)</td><td>4 (2.8)</td><td>3 (2.3)</td></tr><tr><td>Missing</td><td>21 (6.9)</td><td>10 (6.9)</td><td>4 (3.1)</td></tr><tr><td><strong>Parental Socio-Economic Status</strong></td><td></td><td></td><td></td></tr><tr><td><strong>Maternal job at birth (ISCO-1968 codes)</strong></td><td></td><td></td><td></td></tr><tr><td>Not working at time of birth</td><td>111 (36.5)</td><td>61 (42.1)</td><td>48 (37.2)</td></tr><tr><td>Professional, Technical and Related Workers (0/1)</td><td>57 (18.8)</td><td>28 (19.3)</td><td>34 (26.4)</td></tr><tr><td>Administrative and Managerial Workers (2)</td><td>9 (3.0)</td><td>0 (0.0)</td><td>5 (3.9)</td></tr><tr><td>Clerical and Related Workers (3)</td><td>61 (20.1)</td><td>26 (17.9)</td><td>23 (17.8)</td></tr><tr><td>Sales Workers (4)</td><td>14 (4.6)</td><td>1 (0.7)</td><td>5 (3.9)</td></tr><tr><td>Service Workers (5)</td><td>27 (8.9)</td><td>14 (9.7)</td><td>10 (7.8)</td></tr><tr><td>Agricultural, Animal Husbandry and Forestry Workers, Fishermen and Hunters (6)</td><td>8 (2.6)</td><td>4 (2.8)</td><td>1 (0.8)</td></tr><tr><td>Production and Related Workers, Transport Equipment Operators and Labourers (7/8/9)</td><td>17 (5.6)</td><td>11 (7.6)</td><td>3 (2.3)</td></tr><tr><td><strong>Maternal job at birth's industry (NAF-1999 codes)</strong></td><td></td><td></td><td></td></tr><tr><td>Not working at time of birth</td><td>111 (36.5)</td><td>61 (42.1)</td><td>48 (37.2)</td></tr><tr><td>Agriculture, hunting and forestry (A)</td><td>11 (3.6)</td><td>4 (2.8)</td><td>1 (0.8)</td></tr><tr><td>Mining and quarrying (C)</td><td>0 (0.0)</td><td>1 (0.7)</td><td>0 (0.0)</td></tr><tr><td>Manufacturing (D)</td><td>27 (8.9)</td><td>15 (10.3)</td><td>10 (7.8)</td></tr><tr><td>Electricity, gas and water supply (E)</td><td>0 (0.0)</td><td>1 (0.7)</td><td>0 (0.0)</td></tr><tr><td>Construction (F)</td><td>2 (0.7)</td><td>0 (0.0)</td><td>1 (0.8)</td></tr><tr><td>Wholesale and retail trade; repair of motor vehicles, motorcycles and personal and household goods (G)</td><td>16 (5.3)</td><td>4 (2.8)</td><td>10 (7.8)</td></tr><tr><td>Hotels and restaurants (H)</td><td>3 (1.0)</td><td>3 (2.1)</td><td>1 (0.8)</td></tr><tr><td>Transport, storage and communication (I)</td><td>10 (3.3)</td><td>3 (2.1)</td><td>6 (4.7)</td></tr><tr><td>Financial intermediation (J)</td><td>12 (3.9)</td><td>4 (2.8)</td><td>6 (4.7)</td></tr><tr><td>Real estate, renting and business activities (K)</td><td>12 (3.9)</td><td>2 (1.4)</td><td>5 (3.9)</td></tr><tr><td>Public administration and defence; compulsory social security (L)</td><td>14 (4.6)</td><td>12 (8.3)</td><td>4 (3.1)</td></tr><tr><td>Education (M)</td><td>30 (9.9)</td><td>11 (7.6)</td><td>14 (10.9)</td></tr><tr><td>Health and social work (N)</td><td>46 (15.1)</td><td>20 (13.8)</td><td>21 (16.3)</td></tr><tr><td>Other community, social and personal service activities (O)</td><td>10 (3.3)</td><td>1 (0.7)</td><td>2 (1.6)</td></tr><tr><td>Private households with employed persons (P)</td><td>0 (0.0)</td><td>3 (2.1)</td><td>0 (0.0)</td></tr><tr><td><strong>Paternal job at birth (ISCO-1968 codes)</strong></td><td></td><td></td><td></td></tr><tr><td>Not working at time of birth</td><td>42 (13.8)</td><td>20 (13.8)</td><td>11 (8.5)</td></tr><tr><td>Professional, Technical and Related Workers (0/1)</td><td>65 (21.4)</td><td>39 (26.9)</td><td>51 (39.5)</td></tr><tr><td>Administrative and Managerial Workers (2)</td><td>24 (7.9)</td><td>4 (2.8)</td><td>12 (9.3)</td></tr><tr><td>Clerical and Related Workers (3)</td><td>24 (7.9)</td><td>14 (9.7)</td><td>12 (9.3)</td></tr><tr><td>Sales Workers (4)</td><td>13 (4.3)</td><td>6 (4.1)</td><td>8 (6.2)</td></tr><tr><td>Service Workers (5)</td><td>24 (7.9)</td><td>5 (3.4)</td><td>6 (4.7)</td></tr><tr><td>Agricultural, Animal Husbandry and Forestry Workers, Fishermen and Hunters (6)</td><td>21 (6.9)</td><td>11 (7.6)</td><td>2 (1.6)</td></tr><tr><td>Production and Related Workers, Transport Equipment Operators and Labourers (7/8/9)</td><td>89 (29.3)</td><td>46 (31.7)</td><td>27 (20.9)</td></tr><tr><td>Military</td><td>2 (0.7)</td><td>0 (0.0)</td><td>0 (0.0)</td></tr></tbody></table>
<table><caption>**Table 1 (continued)**</caption><thead><tr><th></th><th>TGCT cases (N = 304)</th><th>Group A controls (N = 145)</th><th>Group B controls (N = 129)</th></tr><tr><th></th><th>n (%)</th><th>n (%)</th><th>n (%)</th></tr></thead><tbody><tr><td><strong>Paternal job at birth's industry (NAF-1999 codes)</strong></td><td></td><td></td><td></td></tr><tr><td>Not working at time of birth</td><td>42 (13.8)</td><td>20 (13.8)</td><td>11 (8.5)</td></tr><tr><td>Agriculture,hunting and forestry (A)</td><td>25 (8.2)</td><td>11 (7.6)</td><td>3 (2.3)</td></tr><tr><td>Manufacturing (D)</td><td>55 (18.1)</td><td>30 (20.7)</td><td>25 (19.4)</td></tr><tr><td>Electricity,gas and water supply (E)</td><td>3 (1.0)</td><td>1 (0.7)</td><td>3 (2.3)</td></tr><tr><td>Construction (F)</td><td>28 (9.2)</td><td>12 (8.3)</td><td>10 (7.8)</td></tr><tr><td>Wholesale and retail trade;repair of motor vehicles,motorcycles and personal and household goods (G)</td><td>21 (6.9)</td><td>10 (6.9)</td><td>12 (9.3)</td></tr><tr><td>Hotels and restaurants (H)</td><td>7 (2.3)</td><td>3 (2.1)</td><td>2 (1.6)</td></tr><tr><td>Transport,storage and communication (I)</td><td>28 (9.2)</td><td>15 (10.3)</td><td>12 (9.3)</td></tr><tr><td>Financial intermediation (J)</td><td>12 (3.9)</td><td>3 (2.1)</td><td>5 (3.9)</td></tr><tr><td>Real estate,renting and business activities (K)</td><td>21 (6.9)</td><td>10 (6.9)</td><td>11 (8.5)</td></tr><tr><td>Public administration and defence;compulsory social security (L)</td><td>30 (9.9)</td><td>12 (8.3)</td><td>9 (7.0)</td></tr><tr><td>Education (M)</td><td>16 (5.3)</td><td>12 (8.3)</td><td>12 (9.3)</td></tr><tr><td>Health and social work (N)</td><td>13 (4.3)</td><td>4 (2.8)</td><td>9 (7.0)</td></tr><tr><td>Other community,social and personal service activities (O)</td><td>3 (1.0)</td><td>2 (1.4)</td><td>4 (3.1)</td></tr><tr><td>Extra-territorial organizations and bodies (Q)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>1 (0.8)</td></tr><tr><td><strong>Maternal education</strong></td><td></td><td></td><td></td></tr><tr><td>Baccalaureate's degree or less</td><td>192 (63.2)</td><td>83 (57.2)</td><td>59 (45.7)</td></tr><tr><td>Graduate studies</td><td>84 (27.6)</td><td>35 (24.1)</td><td>52 (40.3)</td></tr><tr><td>Other</td><td>28 (9.2)</td><td>26 (17.9)</td><td>18 (14.0)</td></tr><tr><td>Missing</td><td>0 (0.0)</td><td>1 (0.7)</td><td>0 (0.0)</td></tr><tr><td><strong>Parental incomes at birth</strong></td><td></td><td></td><td></td></tr><tr><td>0 to less than 5000€</td><td>63 (20.7)</td><td>42 (29.0)</td><td>35 (27.1)</td></tr><tr><td>5000 to less than 10,000€</td><td>55 (18.1)</td><td>22 (15.2)</td><td>18 (14.0)</td></tr><tr><td>10,000 to less than 20,000€</td><td>41 (13.5)</td><td>22 (15.2)</td><td>16 (12.4)</td></tr><tr><td>20,000 to less than 30,000€</td><td>21 (6.9)</td><td>5 (3.4)</td><td>13 (10.1)</td></tr><tr><td>30,000€ and more</td><td>24 (7.9)</td><td>5 (3.4)</td><td>5 (3.9)</td></tr><tr><td>Missing</td><td>100 (32.9)</td><td>49 (33.8)</td><td>42 (32.6)</td></tr><tr><td><strong>Residential Characteristics</strong></td><td></td><td></td><td></td></tr><tr><td><strong>Urban status of the place of birth,commune level</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>73 (24.0)</td><td>36 (24.8)</td><td>27 (20.9)</td></tr><tr><td>Yes</td><td>231 (76.0)</td><td>109 (75.2)</td><td>102 (79.1)</td></tr><tr><td><strong>Presence of crops in 500 m buffer around place of birth</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>76 (25.0)</td><td>39 (26.9)</td><td>38 (29.5)</td></tr><tr><td>Yes</td><td>223 (73.4)</td><td>104 (71.7)</td><td>90 (69.8)</td></tr><tr><td>Missing</td><td>5 (1.6)</td><td>2 (1.4)</td><td>1 (0.8)</td></tr><tr><td><strong>Possession of a garden</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>153 (50.3)</td><td>76 (52.4)</td><td>64 (49.6)</td></tr><tr><td>Yes</td><td>151 (49.7)</td><td>69 (47.6)</td><td>65 (50.4)</td></tr><tr><td><strong>Possession of a vegetable garden</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>223 (73.4)</td><td>97 (66.9)</td><td>88 (68.2)</td></tr><tr><td>Yes</td><td>81 (26.6)</td><td>48 (33.1)</td><td>41 (31.8)</td></tr><tr><td><strong>Possession of an orchard</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>261 (85.9)</td><td>127 (87.6)</td><td>110 (85.3)</td></tr><tr><td>Yes</td><td>43 (14.1)</td><td>18 (12.4)</td><td>19 (14.7)</td></tr></tbody></table>
<table><caption>**Table 1** (continued)</caption><thead><tr><th></th><th>TGCT cases (N = 304)</th><th>Group A controls (N = 145)</th><th>Group B controls (N = 129)</th></tr><tr><th></th><th>n (%)</th><th>n (%)</th><th>n (%)</th></tr></thead><tbody><tr><td><strong>Gardening activities during the spring/summer seasons</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>230 (75.7)</td><td>102 (70.3)</td><td>92 (71.3)</td></tr><tr><td>Yes</td><td>74 (24.3)</td><td>42 (29.0)</td><td>37 (28.7)</td></tr><tr><td>Missing</td><td>0 (0.0)</td><td>1 (0.7)</td><td>0 (0.0)</td></tr><tr><td><strong>Possession of pets</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>69 (22.7)</td><td>32 (22.1)</td><td>29 (22.5)</td></tr><tr><td>Yes</td><td>235 (77.3)</td><td>113 (77.9)</td><td>100 (77.5)</td></tr><tr><td><strong>Possession of indoor plants</strong></td><td></td><td></td><td></td></tr><tr><td>No</td><td>178 (58.6)</td><td>81 (55.9)</td><td>78 (60.5)</td></tr><tr><td>Yes</td><td>126 (41.4)</td><td>64 (44.1)</td><td>51 (39.5)</td></tr></tbody></table>

<table><caption>**Table 2** Distribution of TGCT cases, overall and by subtypes, and group A and group B controls by age at diagnosis/inclusion (years), case-control study, N = 578, France, 2015–2018</caption><thead><tr><th>Age at diagnosis/<br>inclusion (years)</th><th>Group A controls<br>n (%)</th><th>Group B controls<br>n (%)</th><th>TGCT cases<sup>a</sup><br>n (%)</th><th>Non-seminoma cases<br>n (%)</th><th>Seminoma cases<br>n (%)</th></tr></thead><tbody><tr><td>&lt;=25</td><td>7 (4.8)</td><td>5 (3.9)</td><td>54 (17.8)</td><td>35 (26.5)</td><td>13 (9.0)</td></tr><tr><td>26–30</td><td>23 (15.9)</td><td>28 (21.7)</td><td>79 (26.0)</td><td>38 (28.8)</td><td>32 (22.2)</td></tr><tr><td>31–35</td><td>63 (43.4)</td><td>55 (42.6)</td><td>84 (27.6)</td><td>30 (22.7)</td><td>44 (30.6)</td></tr><tr><td>36–40</td><td>36 (24.8)</td><td>32 (24.8)</td><td>59 (19.4)</td><td>20 (15.2)</td><td>38 (26.4)</td></tr><tr><td>&gt;=41</td><td>16 (11.0)</td><td>9 (7.0)</td><td>28 (9.2)</td><td>9 (6.8)</td><td>17 (11.8)</td></tr><tr><td>Total</td><td>145 (100.0)</td><td>129 (100.0)</td><td>304 (100.0)</td><td>132 (100.0)</td><td>144 (100.0)</td></tr></tbody></table>

CI = 0.72–1.79 and OR = 0.74, CI = 0.39–1.39, respectively), while a statistically significant increase in NS risk was observed for indirect outdoor use of pesticides (OR = 2.33, CI = 1.24–4.39) (Table S6).

There was no effect modification by birth cohorts and no association was observed overall (Table S7). We found no effect modification by status of birthplace and no association overall (Table S8). Neither effect modification nor associations were observed when stratifying on the season during which the first trimester of pregnancy occurred (Table S9). Among mothers/relatives who reported doing gardening activities, the use of pesticides in gardening by another person sharing the household was 30.1% (N = 46/153), while it was 9.7% (N = 41/424) among those who did not (Table S10). Although there was no effect modification by reported gardening activities (p-for-interaction = 0.63), the use of pesticides during gardening by another person sharing the household was positively associated with TGCT and NS in particular among mothers/relatives who

reported not doing gardening activities (OR = 2.30, CI = 1.06–4.96, and OR = 4.75, CI = 1.83–12.34, respectively); there was no association among mothers/relatives who reported doing gardening activities.

Excluding TGCT cases with personal history of cryptorchidism did not modify our results; a positive OR was observed for the domestic use of fungicides (OR = 1.78, CI = 1.06–2.99) (Table S11). In the subpopulation restricted to participants with no family history of cryptorchidism and no family history of testicular cancer, our results were similar to the main results. We observed statistically significant positive associations with TGCT for the domestic use of fungicides (OR = 1.92, CI = 1.12–3.30) (Table S11). Excluding TGCT cases not confirmed by pathology reports did not change our results (Table S11). The associations with domestic use of fungicides remained statistically significant, with OR for TGCT of 1.68 (CI = 1.00–2.83). Restricting the analyses to participants with interviewed mothers only did not change

<footnote><sup>a</sup> Include N = 1 confirmed TGCT case of regressed germ cell tumours (GONIS-related TGCT, unknown subtype) and N = 27 TGCT cases not confirmed by pathology report. As the median delay between date of diagnosis and date of inclusion among the cases confirmed by pathology report was 8 days, we used age at inclusion instead of age at diagnosis for the 27 cases not confirmed by pathology report</footnote>
<table><caption>**Table 3** Odds ratios and 95% confidence intervals for TGCT associated with domestic use of pesticides during early periods of development, overall and according to histological subtypes, case-control study, N=578, France, 2015–2018</caption><thead><tr><th rowspan="3"></th><th>Total</th><th colspan="2">Controls A + B</th><th colspan="2">All TGCT cases</th><th colspan="2">Non-Seminomas</th><th colspan="2">Seminomas</th><th rowspan="3">P-HET<sup>b</sup></th></tr><tr><th>(N=578)</th><th>(N=274)</th><th colspan="2">(N=304)</th><th colspan="2">(N=132)</th><th colspan="2">(N=144)</th></tr><tr><th>n (%)</th><th>n (%)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th></tr></thead><tbody><tr><td><strong>Domestic use of insecticides</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.12</td></tr><tr><td>No</td><td>129 (22.3)</td><td>62 (22.6)</td><td>67 (22)</td><td>1.00</td><td>28 (21.2)</td><td>1.00</td><td>34 (23.6)</td><td>1.00</td><td></td><td></td></tr><tr><td>Yes</td><td>447 (77.3)</td><td>211 (77.0)</td><td>236 (77.6)</td><td>1.27 (0.80, 2.01)</td><td>104 (78.8)</td><td>1.78 (0.91, 3.48)</td><td>109 (75.7)</td><td>0.89 (0.50, 1.59)</td><td></td><td></td></tr><tr><td>Missing</td><td>2 (0.3)</td><td>1 (0.4)</td><td>1 (0.3)</td><td>–</td><td>0 (0.0)</td><td>–</td><td>1 (0.7)</td><td>–</td><td></td><td></td></tr><tr><td><strong>Domestic use of fungicides</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.08</td></tr><tr><td>No</td><td>478 (82.7)</td><td>234 (85.4)</td><td>244 (80.3)</td><td>1.00</td><td>100 (75.8)</td><td>1.00</td><td>123 (85.4)</td><td>1.00</td><td></td><td></td></tr><tr><td>Yes</td><td>92 (15.9)</td><td>37 (13.5)</td><td>55 (18.1)</td><td>1.73 (1.04, 2.87)</td><td>31 (23.5)</td><td>2.44 (1.26, 4.74)</td><td>18 (12.5)</td><td>1.04 (0.52, 2.07)</td><td></td><td></td></tr><tr><td>Missing</td><td>8 (1.4)</td><td>3 (1.1)</td><td>5 (1.6)</td><td>–</td><td>1 (0.8)</td><td>–</td><td>3 (2.1)</td><td>–</td><td></td><td></td></tr><tr><td><strong>Domestic use of herbicides</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.17</td></tr><tr><td>No</td><td>508 (87.9)</td><td>242 (88.3)</td><td>266 (87.5)</td><td>1.00</td><td>111 (84.1)</td><td>1.00</td><td>129 (89.6)</td><td>1.00</td><td></td><td></td></tr><tr><td>Yes</td><td>70 (12.1)</td><td>32 (11.7)</td><td>38 (12.5)</td><td>1.15 (0.67, 2.00)</td><td>21 (15.9)</td><td>1.64 (0.83, 3.27)</td><td>15 (10.4)</td><td>0.80 (0.37, 1.71)</td><td></td><td></td></tr><tr><td>Missing</td><td>0 (0.0)</td><td>0 (0)</td><td>0 (0.0)</td><td>–</td><td>0 (0.0)</td><td>–</td><td>0 (0.0)</td><td>–</td><td></td><td></td></tr><tr><td><strong>Domestic use of any pesticides</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.18</td></tr><tr><td>No</td><td>119 (20.6)</td><td>57 (20.8)</td><td>62 (20.4)</td><td>1.00</td><td>25 (18.9)</td><td>1.00</td><td>32 (22.2)</td><td>1.00</td><td></td><td></td></tr><tr><td>Yes</td><td>456 (78.9)</td><td>215 (78.5)</td><td>241 (79.3)</td><td>1.18 (0.74, 1.88)</td><td>107 (81.1)</td><td>1.56 (0.79, 3.06)</td><td>111 (77.1)</td><td>0.84 (0.47, 1.51)</td><td></td><td></td></tr><tr><td>Missing</td><td>3 (0.5)</td><td>2 (0.7)</td><td>1 (0.3)</td><td>–</td><td>0 (0.0)</td><td>–</td><td>1 (0.7)</td><td>–</td><td></td><td></td></tr></tbody></table>

OR odds ratios, CI confidence intervals

the observed associations with domestic use of fungicides (OR = 1.82, CI = 1.09–3.03) (Table S11).

Discussion

In this French case-control study, domestic use of fungicides during early periods of development was found positively associated with increased risk of TGCT in the sons, driven by an association with the histological subtype NS, while no association was found for domestic use of insecticides and herbicides. Additional adjustment for age at diagnosis/inclusion did not alter the main conclusion. When focusing on the different household applications, the results also suggest positive associations between risk of NS and use of insecticides on pets and use of fungicides and/or insecticides for woodwork.

Prevalence of domestic use of pesticides was high in the study population and was driven by the use of insecticides (77%); the prevalence of use of fungicides was much lower (16%). Prevalence was similar to that of other studies conducted in France, notably for the observed higher prevalence of insecticide use and much lower prevalence of fungicide use [11, 32, 35, 36], and worldwide [37–39];

although published data covered more recent time periods.

To our knowledge, the only published data on specific pesticides used domestically during the study period are available from the USA [40, 41], Canada [42] and UK [43]. Based on these studies, even if active ingredients reported were not always the same, organophosphorus, pyrethroids, carbamates and acid herbicides pesticides appeared to be most commonly used during the period of interest. Regarding fungicides, the American National Household Pesticide Usage Study (1976–1977) [41] reported that Captan (phthalimide) and Folpet (dicarbomimide) were among the most used pesticides in households during this period. These families were still in use in France in 2010, according to The French Agency for Food, Environmental and Occupational Health & Safety [44]. Organochlorine insecticides, however, disappeared at the turn of the 90’s; and the use of pyrethroids compounds seemed to increase as the use of organophosphorus compounds was decreasing.

No previous study found increased risks of TGCT and NS for the use of fungicides; the main reason being

<footnote><sup>a</sup> Estimates obtained comparing TGCT cases to group A and group B controls combined. Analysis was restricted to subjects with no missing data for the exposure variable (0.2 to 1.4% excluded)</footnote>

<footnote><sup>b</sup> P-value for heterogeneity derived from the Likelihood Ratio Test, comparing seminoma versus non-seminoma tumours</footnote>
<table><caption>**Table 4** Odds ratios and 95% confidence intervals for TGCT associated with domestic use of pesticides during early periods of development, by household applications, overall and according to histological subtypes, case-control study, N = 578, France, 2015–2018</caption><thead><tr><th rowspan="2"></th><th rowspan="2">Total<br>(N = 578)</th><th rowspan="2">Controls A + B<br>(N = 274)</th><th colspan="2">All TGCT cases<br>(N = 304)</th><th colspan="2">Non-Seminomas<br>(N = 132)</th><th colspan="2">Seminomas<br>(N = 144)</th><th rowspan="2">P-HET<sup>b</sup></th></tr><tr><th>n (%)</th><th>n (%)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th></tr></thead><tbody><tr><td colspan="10"><strong>For Gardening</strong></td></tr><tr><td colspan="10"><strong>Use of pesticides</strong></td></tr><tr><td>No</td><td>433 (74.9)</td><td>206 (75.2)</td><td>227 (74.7)</td><td>1.00</td><td>90 (68.2)</td><td>1.00</td><td>116 (80.6)</td><td>1.00</td><td rowspan="3">0.08</td></tr><tr><td>Yes</td><td>138 (23.9)</td><td>65 (23.7)</td><td>73 (24.0)</td><td>1.18 (0.77, 1.82)</td><td>39 (29.5)</td><td>1.60 (0.92, 2.78)</td><td>27 (18.8)</td><td>0.78 (0.43, 1.40)</td></tr><tr><td>Missing</td><td>7 (1.2)</td><td>3 (1.1)</td><td>4 (1.3)</td><td>-</td><td>3 (2.3)</td><td>-</td><td>1 (0.7)</td><td>-</td></tr><tr><td colspan="10"><strong>Use of insecticides</strong></td></tr><tr><td>No</td><td>509 (88.1)</td><td>240 (87.6)</td><td>269 (88.5)</td><td>1.00</td><td>114 (86.4)</td><td>1.00</td><td>130 (90.3)</td><td>1.00</td><td rowspan="3">0.17</td></tr><tr><td>Yes</td><td>69 (11.9)</td><td>34 (12.4)</td><td>35 (11.5)</td><td>1.11 (0.63, 1.94)</td><td>18 (13.6)</td><td>1.66 (0.83, 3.33)</td><td>14 (9.7)</td><td>0.79 (0.36, 1.73)</td></tr><tr><td>Missing</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td></tr><tr><td colspan="10"><strong>Use of herbicides</strong></td></tr><tr><td>No</td><td>508 (87.9)</td><td>242 (88.3)</td><td>266 (87.5)</td><td>1.00</td><td>111 (84.1)</td><td>1.00</td><td>129 (89.6)</td><td>1.00</td><td rowspan="3">0.17</td></tr><tr><td>Yes</td><td>70 (12.1)</td><td>32 (11.7)</td><td>38 (12.5)</td><td>1.15 (0.67, 2.00)</td><td>21 (15.9)</td><td>1.64 (0.83, 3.27)</td><td>15 (10.4)</td><td>0.80 (0.37, 1.71)</td></tr><tr><td>Missing</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td></tr><tr><td colspan="10"><strong>Use of fungicides</strong></td></tr><tr><td>No</td><td>541 (93.6)</td><td>257 (93.8)</td><td>284 (93.4)</td><td>1.00</td><td>122 (92.4)</td><td>1.00</td><td>135 (93.8)</td><td>1.00</td><td rowspan="3">0.75</td></tr><tr><td>Yes</td><td>37 (6.4)</td><td>17 (6.2)</td><td>20 (6.6)</td><td>1.22 (0.59, 2.54)</td><td>10 (7.6)</td><td>1.28 (0.50, 3.30)</td><td>9 (6.3)</td><td>1.03 (0.40, 2.65)</td></tr><tr><td>Missing</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td></tr><tr><td colspan="10"><strong>On Indoor Plants<sup>c</sup></strong></td></tr><tr><td colspan="10"><strong>Use of insecticides</strong></td></tr><tr><td>No</td><td>545 (94.3)</td><td>262 (95.6)</td><td>283 (93.1)</td><td>1.00</td><td>124 (93.9)</td><td>1.00</td><td>133 (92.4)</td><td>1.00</td><td rowspan="3">0.82</td></tr><tr><td>Yes</td><td>28 (4.8)</td><td>10 (3.6)</td><td>18 (5.9)</td><td>2.06 (0.89, 4.75)</td><td>7 (5.3)</td><td>1.90 (0.62, 5.80)</td><td>10 (6.9)</td><td>2.26 (0.83, 6.15)</td></tr><tr><td>Missing</td><td>5 (0.9)</td><td>2 (0.7)</td><td>3 (1.0)</td><td>-</td><td>1 (0.8)</td><td>-</td><td>1 (0.7)</td><td>-</td></tr><tr><td colspan="10"><strong>For Pest Control</strong></td></tr><tr><td colspan="10"><strong>Use of insecticides</strong></td></tr><tr><td>No</td><td>174 (30.1)</td><td>82 (29.9)</td><td>92 (30.3)</td><td>1.00</td><td>38 (28.8)</td><td>1.00</td><td>45 (31.3)</td><td>1.00</td><td rowspan="3">0.19</td></tr><tr><td>Yes</td><td>404 (69.9)</td><td>192 (70.1)</td><td>212 (69.7)</td><td>1.12 (0.75, 1.69)</td><td>94 (71.2)</td><td>1.49 (0.83, 2.67)</td><td>99 (68.8)</td><td>0.89 (0.54, 1.49)</td></tr><tr><td>Missing</td><td>0 (0.0)</td><td>0 (0.0)</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td><td>0 (0.0)</td><td>-</td></tr><tr><td colspan="10"><strong>Insecticide use<br>frequency against<br>crawling insects</strong></td></tr><tr><td>No use</td><td>371 (64.2)</td><td>183 (66.8)</td><td>188 (61.8)</td><td>1.00</td><td>79 (59.8)</td><td>1.00</td><td>93 (64.6)</td><td>1.00</td><td rowspan="5">0.58</td></tr><tr><td>Less than once a<br>month</td><td>153 (26.5)</td><td>67 (24.5)</td><td>86 (28.3)</td><td>1.31 (0.85, 2.02)</td><td>40 (30.3)</td><td>1.65 (0.90, 3.04)</td><td>36 (25.0)</td><td>1.07 (0.62, 1.86)</td></tr><tr><td>Less than once a<br>week</td><td>32 (5.5)</td><td>17 (6.2)</td><td>15 (4.9)</td><td>0.83 (0.38, 1.80)</td><td>7 (5.3)</td><td>0.98 (0.35, 2.73)</td><td>8 (5.6)</td><td>0.73 (0.29, 1.88)</td></tr><tr><td>More than once a<br>week to daily</td><td>20 (3.5)</td><td>6 (1.8)</td><td>14 (3.3)</td><td>1.78 (0.56, 5.60)</td><td>5 (3.8)</td><td>1.43 (0.27, 7.68)</td><td>7 (4.9)</td><td>2.07 (0.48, 9.00)</td></tr><tr><td>Missing</td><td>2 (0.3)</td><td>1 (0.4)</td><td>1 (0.3)</td><td>-</td><td>1 (0.8)</td><td>-</td><td>0 (0.0)</td><td>-</td></tr><tr><td colspan="10"><strong>P trend</strong></td></tr><tr><td colspan="10"><strong>Insecticide use<br>frequency against flying<br>insects</strong></td></tr><tr><td>No use</td><td>228 (39.4)</td><td>106 (38.7)</td><td>122 (40.1)</td><td>1.00</td><td>51 (38.6)</td><td>1.00</td><td>58 (40.3)</td><td>1.00</td><td rowspan="3">0.33</td></tr><tr><td>Less than once a<br>month</td><td>153 (26.5)</td><td>73 (26.6)</td><td>80 (26.3)</td><td>1.16 (0.73, 1.85)</td><td>36 (27.3)</td><td>1.53 (0.79, 2.96)</td><td>40 (27.8)</td><td>0.93 (0.52, 1.67)</td></tr><tr><td>Less than once a<br>week</td><td>83 (14.4)</td><td>40 (14.6)</td><td>43 (14.1)</td><td>0.94 (0.53, 1.67)</td><td>22 (16.7)</td><td>1.43 (0.67, 3.05)</td><td>16 (11.1)</td><td>0.62 (0.28, 1.36)</td></tr></tbody></table>
<table><caption>**Table 4** (continued)</caption><thead><tr><th rowspan="3"></th><th>Total</th><th>Controls A + B</th><th colspan="2">All TGCT cases</th><th colspan="2">Non-Seminomas</th><th colspan="2">Seminomas</th><th rowspan="3">P-HET<sup>b</sup></th></tr><tr><th>(N = 578)</th><th>(N = 274)</th><th colspan="2">(N = 304)</th><th colspan="2">(N = 132)</th><th colspan="2">(N = 144)</th></tr><tr><th>n (%)</th><th>n (%)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th><th>n (%)</th><th>OR<sup>a</sup> (95% CI)</th></tr></thead><tbody><tr><td>More than once a week</td><td>77 (13.3)</td><td>39 (14.2)</td><td>38 (12.5)</td><td>0.94 (0.52, 1.70)</td><td>16 (12.1)</td><td>1.17 (0.50, 2.76)</td><td>19 (13.2)</td><td>0.87 (0.41, 1.83)</td><td></td></tr><tr><td>Daily</td><td>35 (6.1)</td><td>14 (5.1)</td><td>21 (6.9)</td><td>1.98 (0.89, 4.44)</td><td>7 (5.3)</td><td>1.21 (0.36, 4.10)</td><td>11 (7.6)</td><td>2.60 (0.95, 7.10)</td><td></td></tr><tr><td>Missing</td><td>2 (0.3)</td><td>2 (0.7)</td><td>0 (0.0)</td><td>–</td><td>0 (0.0)</td><td>–</td><td>0 (0.0)</td><td>–</td><td></td></tr><tr><td><strong>P trend</strong></td><td></td><td></td><td></td><td>0.45</td><td></td><td>0.59</td><td></td><td>0.52</td><td></td></tr><tr><td><strong>Wood and Mold<sup>d</sup></strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td><strong>Use of fungicides and/or insecticides for woodwork</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.03</td></tr><tr><td>No</td><td>517 (89.4)</td><td>249 (90.9)</td><td>268 (88.2)</td><td>1.00</td><td>110 (83.3)</td><td>1.00</td><td>134 (93.1)</td><td>1.00</td><td></td></tr><tr><td>Yes</td><td>57 (9.9)</td><td>24 (8.8)</td><td>33 (10.9)</td><td>1.36 (0.73, 2.55)</td><td>21 (15.9)</td><td>2.35 (1.06, 5.20)</td><td>8 (5.6)</td><td>0.58 (0.22, 1.53)</td><td></td></tr><tr><td>Missing</td><td>4 (0.7)</td><td>1 (0.4)</td><td>3 (1.0)</td><td>–</td><td>1 (0.8)</td><td>–</td><td>2 (1.4)</td><td>–</td><td></td></tr><tr><td><strong>Pets</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>0.01</td></tr><tr><td><strong>Use of insecticides on cats and dogs</strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td>410 (70.9)</td><td>195 (71.2)</td><td>215 (70.7)</td><td>1.00</td><td>85 (64.4)</td><td>1.00</td><td>114 (79.2)</td><td>1.00</td><td></td></tr><tr><td>Yes</td><td>163 (28.2)</td><td>76 (27.7)</td><td>87 (28.6)</td><td>1.14 (0.76, 1.71)</td><td>45 (34.1)</td><td>1.95 (1.12, 3.40)</td><td>30 (20.8)</td><td>0.66 (0.38, 1.15)</td><td></td></tr><tr><td>Missing</td><td>5 (0.9)</td><td>3 (1.1)</td><td>2 (0.7)</td><td>–</td><td>2 (1.5)</td><td>–</td><td>0 (0.0)</td><td>–</td><td></td></tr><tr><td><strong>Use of insecticides on other animals<sup>e</sup></strong></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>No</td><td>565 (97.8)</td><td>268 (97.8)</td><td>297 (97.7)</td><td>1.00</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Yes</td><td>11 (1.9)</td><td>6 (2.2)</td><td>5 (1.6)</td><td>0.38 (0.09, 1.61)</td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>Missing</td><td>2 (0.3)</td><td>0 (0.0)</td><td>2 (0.7)</td><td>–</td><td></td><td></td><td></td><td></td><td></td></tr></tbody></table>

OR odds ratios, CI confidence intervals

however that fungicides were not specifically investigated in other studies. In contrast to our results, an increased risk of TGCT was observed for self-reported household use of insecticides (OR=3.23, CI=1.15–9.11) [13]. In addition, positive associations have been found for gardening tasks, with OR for TGCT of 1.84 (CI=1.23–2.75) in a French case-control study [15], and for the use of pesticides in the garden, with OR for TGCT from 1.83 (CI 1.02–3.29) to 4.80 (CI=0.91–25.30) and OR for NS of 2.54 (CI=1.26–5.11) in Italian settings [13, 14]. However, these studies covered adulthood exposure periods, and part of these results did not remain statistically significant in multivariate models. Moreover, it is noteworthy that cryptorchidism may be an unnecessary adjustment in these studies in light of the current understanding of the TDS [7]. In our study, controls with personal history of cryptorchidism were not eligible. Moreover, because cases and controls with personal and/or family history

of cryptorchidism and testicular cancer may be at higher risk of developing TGCT, they were excluded in sensitivity analyses, and the previously observed associations remained.

There was no heterogeneity between birth cohorts and urban/rural status of birthplace. Results were obtained with a large uncertainty because of small numbers and we cannot exclude that they were due to chance. No clear findings have been reported on the association between living in a rural environment and risk of TGCT; heterogeneity in the definition of rural areas might be a reason [8, 14, 15].

Our analyses showed a positive association for the risk of NS with the use of pesticides for gardening by another person than the mother/relative sharing the same household (most likely to be the cases’/controls’ father although this has not been investigated), and this was particularly observed among mothers who had not reported any

<footnote><sup>a</sup> Estimates obtained comparing TGCT cases to group A and group B controls combined. Analysis was restricted to subjects with no missing data for the exposure variable (0.2 to 1.4% excluded)</footnote>

<footnote><sup>b</sup> P-value for heterogeneity derived from the Likelihood Ratio Test, comparing seminoma versus non-seminoma tumours</footnote>

<footnote><sup>c</sup> Use of fungicides on indoors plants was not presented because some cells had less than 5 subjects</footnote>

<footnote><sup>d</sup> Use of fungicides on mold was not presented because some cells had less than 5 subjects</footnote>

<footnote><sup>e</sup> Analysis by subtype for use of insecticides on other animals was not presented because some cells had less than 5 subjects</footnote>
gardening activities. While this observation might suggest a possible role of indirect maternal exposures, it might also support the hypothesis of a potential role of paternal exposures during early periods of development. Exposures to endocrine disrupting chemicals (EDCs), especially during preconception, have been suggested to cause epigenetic changes in the male germline, which can persist and alter embryonic development and further affect birth outcomes, the reproductive system and genital development [17]. However, this result might also be due to chance and more research is needed to confirm the role of paternal exposures during preconception.

Looking at early periods of development, parental occupational pesticide exposure before birth was not found associated with risk of TGCT in the offspring in a large register-based case-control study conducted in the Nordic countries, although the job-exposure matrix used only considered farmers as exposed to pesticides and there was no detail on the type of pesticides (insecticides, herbicides, fungicides) [45]. In Denmark, however, an increased OR for TGCT was observed for paternal wood-related occupations with potential exposure to wood preservatives that contain insecticides and fungicides (OR = 1.50, CI = 1.01–2.24) [46]. Furthermore, parental domestic exposure to pesticides during pregnancy has been found positively associated with hypospadias [47] and cryptorchidism [48]; however results are currently not conclusive. Although we intended to cover early periods of development, we were unable to clearly distinguish between preconception, pregnancy, birth and early infancy exposures, and in particular the first trimester of pregnancy with respect to testicular development [19]. In an attempt to focus on this specific window, we were able to stratify on the season during which the first trimester of pregnancy occurred, and no statistically significant difference in the risk of TGCT was observed between summer/spring and autumn/winter.

Our findings showed stronger associations for NS than SE; overall, no association was found for SE, regardless of household applications and types of pesticides. Given the earlier onset of NS compared to SE (median age at diagnosis of 25 versus 35 years), it has been hypothesized that perinatal factors may be more relevant for NS than SE [49]. However, the available literature rather reports that SE and NS share important etiologic factors [50, 51]. Also, due to the low numbers and large uncertainty for some of our risk estimates, other studies are warranted to confirm these observations.

The presence of GCNIS cells and increasing understanding of molecular mechanisms support the hypothesis of a developmental origin of TGCT with disruption of primordial germ cell differentiation. Exposure to EDCs has been suggested to cause intrauterine hormonal

imbalance through impairing androgen signalling or mimicking oestrogen signalling, leading to alteration of foetal gonadal development and negative health outcomes in the offspring [5, 9, 52]. In the testes, experimental studies have shown that fungicides can inhibit testicular cytochrome P450 activity – responsible for the detoxication and bioactivation of drugs, carcinogens and pesticides –, induce oxidative damages in the testes leading to apoptosis as well as anti-androgenic effects in male rats [53–55]. Transgenerational epimutations of primordial germ cells were observed following exposure to vinclozolin fungicide [56]. Insecticide and herbicide compounds have shown similar endocrine disruption activities in the testes [57, 58]. In epidemiological studies, no clear association between elevated maternal serum concentrations of hexachlorobenzene fungicide and TGCT risk has been found so far [59, 60], while increased risks were observed for p,p'-dichlorodiphenyldichloroethylene and chlordane organochlorine insecticides; although serum was collected around son's diagnosis [59, 61, 62]. In our study population, users of fungicides were also likely users of insecticides and/or herbicides. Therefore, we cannot surely differentiate an independent effect of fungicides compare to insecticides and herbicides on TGCT risk. Moreover, there might be combined or synergistic effects of pesticides compounds, which we were not able to show with our data. A large number of insecticides, herbicides and fungicides compounds acting as EDCs remain to be investigated in relation to TGCT occurrence, and future research should focus on early periods of development and domestically used compounds [9, 63].

To our knowledge, this is the first study investigating the domestic use of pesticides during early periods of development in association with TGCT occurrence in young men. Strengths of the study include the prospective and multicentric recruitment of cases and controls, covering the whole Metropolitan French territory, and the inclusion of participants' mothers/relatives who provided information specific to preconception, pregnancy and early infancy periods [22]. Interviews were conducted by trained investigators blinded to the case-control status of participants and participants' mothers, and contained detailed questions on the domestic use of pesticides, covering several types of pesticides and household applications. We were able to estimate TGCT risk according to the subtypes NS and SE. The large number of pre- and perinatal covariates collected, covering most of established and suspected TGCT risk factors, allowed us to assess confounding, and our results were similar with or without adjustment for these covariates. We were able to take into account other sources of pesticide exposure, such as parental occupation at birth and presence of
crops around residence at birth, for which adjustment did
not change the results.

Our study has several limitations. First, inherent in
case-control studies relying on self-reported information,
reporting bias (accuracy in general) and recall bias (dif-
ferent reporting by case-control status) is of concern, the
latter possibly due to over-reporting of cases or under-
reporting of controls or their mothers. Notably, partici-
pation of mothers was higher among cases than controls
(64% versus 40%). Over-reporting among cases or under-
reporting among controls would both lead to an overesti-
mation of our effect estimates [64]. The use of handouts
and structured questionnaires, and telephone inter-
views blinded to the case-control status were neverthe-
less intended to minimize recall bias. Moreover, intense
recall bias would probably have resulted in generalized
increased prevalence among cases and ORs – which was
not observed here – and not to the association observed
specifically with fungicides. Because participants’ moth-
ers were asked to remember their use of pesticides up to
45 years ago, random reporting error is an issue, possi-
bly leading to an attenuation of any true effect. For very
few participants, a close relative was interviewed. As
the recall of exposures during pregnancy and the peri-
natal period may be less accurate in relatives compared
to mothers, a sensitivity analysis restricting the analyses
to mothers interviewed was also performed, showing no
change in the observed associations. Further, it would
have been difficult to accurately collect more detailed
information, such as the quantity of pesticides applied
or the specific active ingredients used. The retrospective
assessment of pesticide uses during critical time windows
such as before pregnancy and after birth is challenging,
and its validity can be of concern, particularly when the
time lag between exposure and reporting is large. Stud-
ies have shown moderate to high reproducibility for
self-reported past pesticides uses in epidemiological set-
tings, in particular for general pesticide categories (insec-
ticides, herbicides and fungicides), based on repeated
interviews or correlations with pesticide concentration in
dust. Moreover, reliability has been found similar among
cases and controls suggesting that differential recall was
likely to be minimal [65–68]. In our study, self-reporting
was the only means for retrospectively assessing perina-
tal domestic use of pesticides – other methods such as
biomarkers or measurements of pesticide concentration
were not feasible. Our questionnaire collected informa-
tion for the three major types of pesticides (insecticides,
herbicides and fungicides), and the different situations
they could be used at home. Frequency of use and user
(mother or another person in the household) were also
collected for some items. We intended to reduce report-
ing errors by training of interviewers, and thorough

definitions and explanations available for each question-
naire item.

Low response rates are a major challenge to epide-
miological research. The characteristics of the target
population of the present study (i.e. young adult men)
are particularly associated with low response rate in the
literature [69, 70], making it difficult to obtain an unbi-
ased random sample of controls from the general popula-
tion. As testicular cancer and reproduction are sensitive
topics, as well as to minimize non-response bias, and
increase consent to blood sampling, the study was con-
ducted in the University Hospitals, with similar catch-
ment populations for cases, controls A and controls
B, which was the approach that was the most promis-
ing one resulting from our pilot study [23]. The high
response rates of eligible subjects to whom the study
was proposed is similar to previous studies [14, 71, 72].
The non-participants were more likely younger. Also, in
the non-participants the proportion of employment in
intermediate occupations was higher and the propor-
tion of “not professionally active” was lower than among
the subjects that agreed to participate in the study (23%
versus 11, and 18% versus 56%, respectively, data not
shown). Case/controls status of non-respondents was
unfortunately not available. As we cannot exclude in the
clinical setting some underreporting of eligible cases and
controls not interested in participating, the response rate
is likely to be overestimated. Yet, the financial compensa-
tion for participation in interview and blood sampling, as
well as the reimbursement per enrolled participant to the
investigating centers of the staff costs for recruitment,
may have facilitated recruitment, and resulted in a higher
response rate compared to a previous French hospital-
based case-control study on risk factors of TGCT with
cases recruited in CECOS for sperm cryopreservation
and controls in maternity clinics of the same University
hospitals [15]. This study conducted between 2002 and
2005, included 81% of the eligible TGCT cases to whom
the study was proposed, and 39% of controls.

Another main limitation of our study is our inability to
ascertain whether the controls are representative for the
source population from which the cases originate. The
ascertainment protocol recruits controls from hospitals
where the men attend for particular purposes [22, 23].
Hence, they may differ in relevant criteria from men in
the underlying source population not attending the hos-
pital for the same purposes. Whether domestic use of
pesticides is among those is rather speculative. Moreo-
ver, recruitment of controls was performed in centers
with regional scale and activity to avoid over-matching
of cases and controls. Also, the choice of fecund controls
allowed minimizing the risk of recruiting controls with
fertility disorders that may originate in the TDS [7]. To
be noted, that the two independently collected groups of controls did not differ in terms of early life characteristics so that they could be combined to increase the sample size. While the response rate of mothers for which the participating sons agreed to give a contact was good (640 of the 853 subjects contacted, i.e. 75%), the two-stage recruitment process of case/control mothers/relatives in the present study resulted overall in a moderate participation rate of mothers/relatives (50% of the total sample). Very few published studies on TGCT risk factors included case and control mothers. The participation rate of mothers was similar to that of previous studies having included case and control mothers [14, 71–73] and the approach that we used was the one identified as the most promising from the experience of our pilot study [23]. Some differences were observed when comparing participants’ characteristics in groups of interviewed and not interviewed mothers: there were more cases in the interviewed mothers’ group, consequently, the age distribution was different between the two groups and family history of TGCT was reported more frequently in the interviewed mothers’ groups. However, pre- and perinatal factors of participants, and socio-economic statuses of mothers at birth and of participants at interview were similar in the two groups.

Finally, our results may be limited in terms of statistical power, because of the low prevalence for specific exposure variables and small numbers in subgroup analysis, leading to effect estimates with large statistical uncertainty. Despite consistency in main and secondary analyses, we also cannot rule out that our findings were chance due to multiple testing.

## Conclusions

Early life exposures to environmental factors acting as EDC, including pesticides, have been suspected to increase TGCT risk later in life. Our study confirmed that in France parental domestic use of pesticides during early life periods was very common in the general population. Although recall bias is likely, our study suggests that domestic use of fungicides, use of fungicides and/or insecticides for woodwork and use of insecticides on pets during early periods of development may increase the risk of adult TGCT and NS in the offspring. Investigating EDC exposures from multiple sources and during critical periods of development, including preconception, pregnancy and puberty, may be most relevant for future research on TGCT occurrence.

## Abbreviations

ART: Assisted reproduction treatment; CECOS: Centres d'étude et de conservation des oeufs et du sperme; CI: Confidence interval; DDE: p,p'-dichlorodiphenyldichloroethylene; EDC: Endocrine disrupting chemical; GIS: Geographic

Information System; IARC: International Agency for Research on Cancer; ICD-O: International Classification of Disease for Oncology; ISCO: International Standard Classification of Occupations; GCNIS: Germ cell neoplasia in situ; HCB: Hexachlorobenzene; NAF: Nomenclature d'activités française; NS: Non-seminomas; OR: Odds ratio; SAS: Statistical Analysis System; SE: Seminomas; TGCT: Testicular germ cell tumours; WHO: World Health Organization.

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s12940-021-00795-y.

**Additional file 1.** Supplemental Excel File (Tables S1 to S11 and Figure S1).

## Acknowledgments

We gratefully acknowledge the participants and their mothers for providing data, and medical practitioners for providing pathology reports. We are grateful to all medical teams of each CECOS from the French national network *Fédération Française des CECOS*, ART centers and maternity clinics that were involved in recruiting our participants and their mothers: Céline Chalas, Vanessa Gayet, Paul Pirtea, Pietro Santulli, Aurélie Vincent, Edouard Lecarpentier, François Goffinet, Dominique De Ziegler, Khaled Pocate, Virginie Barraud-Lange, Jean-Philippe Wolf, Emmanuel Dulioust, Nathalie Le Foll, Jacques Auger, Anne-Sophie Gille, Laurianne Kremer, Myriam Virlouvet, Lucile Ferreux, Guillemette Perier, Pauline Peretout, Diane Rivet and Sandrine Rulle (Cochin Hospital, Paris); Rachel Levy, Nathalie Sermondade, Yassine Belaid, Marine Durand and Charlène Harbemont (Jean Verdier Hospital, Paris); Xavier Pollet-Villard, Vanina De Larouziere, Laurence Levy-Dutel and Isabelle Berthaut (Tenon Hospital, Paris); Jacqueline Saias-Magnan, Catherine Metzler-Guillemain, Carole Daoud-Deveze and Laurent Nasca (La Conception Hospital, Marseille); Myriam Daudin, Nathalie Moinard, François Isus, Célia Bettiol and Laure Connan from Paule de Viguier Hospital in Toulouse; Laurent Janny Valérie Bruhat and Cyril Bouche (Estaing Hospital, Clermont-Ferrand); Isabelle Koscinski, Marius Teletin, Laëtitia Ladureau-Fritsch, Cécile Greze, Françoise Schmitt, and Charles Pax (Obstetric medico-surgical center, Strasbourg); Aline Papaxanthos, Clément Jimenez, Volcy Soula, Lucie Chansel and Olivier Delorme (Pellegrin maternity Hospital, Bordeaux); Sandrine Giscard d'Estaing, Pascale Dehee and Delphine Yalcinkaya (Femme-Mère-Enfant Hospital, Lyon); Céline Bouillon, Fabrice Guerif, Cynthia Frapsauce, Marie-Laure Couet, Véronique Ract, Olivia Gervereau, Elodie Poisson, Michel Lanoue, Anne Viallon and Catherine Guerin (Bretonneau Hospital, Tour); Bérengère Ducrocq, Julie Guitton, Marie Lefort and Valérie Mitchell (Calmette Hospital, Lille); Marie-Ange Clarotti, Ethel Szman, Amélie Ancelle, Catherine Muris, Corinne Fourmy-Chatel, Christine Denoual-Ziad, Claire De Vienne, Cécile Delesalle, Jean-Paul Bouiller and Antoine Clergeau (Caen Hospital, Caen); Oxana Blagosklonov, Alphée Baily and Séverine Bey (Jean Minjoz Hospital, Besançon); Célia Ravel, Guilhem Jouve, Ségolène Veuà, Laurent Vandenbroucke and Agnès Letremy (South Hospital, Rennes); Patricia Fauque and Julie Barberet (Dijon Hospital, Dijon); Stéphanie Lattes, Emmanuelle Thibault, Fabienne Bernardin, Pierre Besnier and Clémence Martin (l'Archet Hospital, Nice); Catherine Diligent, Françoise Touati, Nicolas Monnin and Christel Hersant (regional University maternity Hospital, Nancy); Vanessa Loup-Cabaniols, Alice Ferrieres, Anna Gala, Elodie Scalici, Lucile Sablayrolles, Tiffany Mullet, Audrey Chabert, Christelle Saintpeyre, Mélanie Caro, Michèle Nou and Marie Sicard (Arnaud de Villeneuve Hospital, Montpellier); Sylvianne Hennebicq, Pascale Hoffmann, Claire Thomas-Cadi, Nicole Quenard, Evelyne Warembourg, Laure Villaret and Julien Bessonnat (Couple-Enfant Hospital, Grenoble); Marie-Claude Blocquaux, Frédérique Carre-Pigeon, Béatrice Delepine, Olivier Graesslin and Julie Burette (Maison Blanche Hospital, Reims). We acknowledge the *Direction de la Recherche Clinique* from the Toulouse University hospital for their contribution to the coordination of the investigation centers. We thank the IPSOS Company for conducting telephone interviews. We acknowledge Charlotte Carretero, Etienne Durand and Bénédicte Thomas who worked on the assessment of historical aerial images using the Gouramic software.

## Disclaimer

Where authors are identified as personnel of the International Agency for Research on Cancer/World Health Organization, the authors alone are
responsible for the views expressed in this article and they do not necessar-
ily represent the decisions, policy or views of the International Agency for
Research on Cancer/World Health Organization.

Authors' contributions

Conception and design of the study: OP, EF, RB, HB, AF, LBu, VD, FE, IB, JP, FB, BC, JS and BF. Acquisition of data: AMD, OP, EF, RB, HB, EB, LG, MD, JS, AF, LB, VD, FE, IB, JP, FB, BC, JS and BF. Analysis of data: AMD, AC. Interpretation of data: AMD, OP, AC, JS, LBu, BC, JS and BF. Drafting of manuscript: AMD, JS and BF. Substantial revising of the drafted manuscript: OP, AC, RB, LG, LBo, LBu, JS and BF. All authors read and approved the final manuscript.

Funding

The project was supported by fundings from the French National Cancer Insti-
tute (INCa, N°2013-143) and the *Institut National de la Santé et de la Recherche
Médicale* (Inserm, N°ENV201306/CLB).

Availability of data and materials

The datasets used and/or analysed during the current study are available from
the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The study received ethical approval from the French Ethics Committee (ref. no. A14-94), the French national agency for medicines and health products safety (ref. no. 140184B-12) and the IARC Ethics Committee (ref. no. 14-26), and was declared to the Commission nationale Informatique et Libertés (MR-001, ref. no. 2016-177). All participants and participants' mothers provided written informed consent.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Environment and Lifestyle Epidemiology Branch, International Agency for Research on Cancer/World Health Organization, 150 cours Albert Thomas, 69372 Lyon, Cedex 08, France. ²Département Prévention, Cancer et Environnement, Centre Léon Bérard, Lyon, France. ³INSERM UMR1296 Radiation: Defense, Health, Environment, Lyon, France. ⁴Université Paris-Saclay, UVSQ, Univ. Paris-Sud, INSERM, Gustave Roussy, Équipe "Exposome et Hérédité", CESP, 94805 Villejuif, France. ⁵IRSET (Institut de Recherche en Santé, Environnement et Travail), UMR S 1085, INSERM, EHESP, CHU Rennes, Rennes University, Rennes, France. ⁶Department of Medical Oncology, Centre Léon Bérard, Lyon, France. ⁷Direction Santé Travail, équipe associée à L'UMRESTTE (UMRT 9405 Université Lyon 1, IFSTTAR), Santé publique France, Lyon, France. ⁸DEFE (Développement Embryonnaire, Fertilité, Environnement) INSERM 1202 Universités Montpellier et Toulouse 3, CECOS Hôpital Paule de Viguier, CHU de Toulouse, Toulouse, France. ⁹Fédération Française des CECOS, Paris, France. ¹⁰CECOS Hôpital Cochin, Paris, France. ¹¹Laboratoire d'Histologie, Biologie de la Reproduction, CECOS Hôpital Tenon, Paris, France. ¹²APHP Sorbonne University, Paris, France. ¹³CNRS, IRD, IMBE, Avignon University, Aix Marseille University, Marseille, France. ¹⁴Centre Clinico-Biologique d'AMP-CECOS, AP-HM La Conception University Hospital, Marseille, France. ¹⁵CHU Clermont-Ferrand, CHU Estaing, AMP, CECOS, Clermont-Ferrand, France. ¹⁶IMOST, INSERM U1240, Faculté Médecine Clermont-Ferrand, Clermont-Ferrand, France. ¹⁷UMRESTTE, UMR T 9405, IFSTTAR, Lyon 1 University, Lyon University, Eiffel University, Lyon, France.

Received: 20 November 2020 Accepted: 7 October 2021

Published online: 28 October 2021

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