# Endpoint study record: Developmental toxicity / ter atogenicity.

OECD 422 Benzenesulfonyl chloride LANXESS - 4120157 9

UUID IUC5-04f31bb6-1c0a-4e53-9182-6570cc948b79

Dossier UUID 0

Author admin / Harlan Laboratories Ltd. / Other / other:

Date 2013-07-26 11:31:37 BST

Remarks



# Administrative Data

Purpose flag robust study summary
Study result type experimental result Study

period The study was performed between 04 October 2012 (formu lation chemistry) and 22 July 2013

(date of final histopathology report). In-life phase of the study was conducted between 17 October 2012 (first day of treatment) and 01 December 2012 (final necropsy)
Reliability 1 (reliable without restriction)

Rationale for reliability incl. deficiencies GLP guideline study.



# Data source

Reference

Reference type Author Year Title Bibliographic

source Testing

laboratory Report

no. Owner

company Company

study no. Report

date
study report Harlan

Laboratories Ltd. 2013 BENZENESULFONYL CHLORIDE

ORAL (GAVAGE) COMBINED REPEAT DOSE TOXICITY STUDY WITH REPRODUCTION/DEVELOPMENTAL TOXICITY SCREENING TEST IN THE RAT (OECD 422). Harlan Laboratories Ltd., Shardlow Business Park, Shardlow, Derbyshire, DE72 2GD UK 41201579 Lanxess Corporation, 111 RIDC Park West Drive, Pittsburgh, PA 15275-1112, USA 2013-07-25



# Materials and methods

Limit test

no 

### Test guideline

Qualifier Guideline Deviations according to other guideline: OECD $22 no 

### GLP compliance

yes (incl. certificate) 

## Test materials

Test material form

other: Liquid 

### Details on test material

Sponsor's identification: Benzenesulfonyl chloride Description: Clear colourless liquid Chemical name: Benzensulphonyl chloride (Benzenesulfonyl chloride) CAS number: 98-09-08 Purity: 99.53% Batch number: KX120302.A Label: Benzenesulphonyl Chloride Lot No KX120302.A Date received: 16 April 2012 Storage conditions: ambient temperature, in the dark Expiry date: 12 April 2013 

## Test animals Species

rat 

### Strain 

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1 of 13 26/07/2013 11:39Wistar



### Details on test animals and environmental conditions

TEST ANIMALS - Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bi cester, Oxon, UK. - Age at study initiation: Approximately twelve wee ks old. - Weight at study initiation: At the start of treatme nt the males weighed 299 to 345 g, the females weigh ed 192 to 226 g. - Fasting period before study: None - Housing: Initially, all animals were housed in gro ups of four in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesa nd Ltd., Cheshire, UK). During the pairing phase, animals were transferred t o polypropylene grid floor cages suspended over trays lined with absorbent paper on a on e male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed indivi dually during gestation and lactation in solid floor polypropylene cages with stai nless steel mesh lids and softwood flakes. - Diet (e.g. ad libitum): A pelleted diet (Rodent 2 018C Teklad Global Certified Diet, Harlan Laboratori es U.K. Ltd., Oxon, UK) was available ad libitum. - Water (e.g. ad libitum): Mains drinking water was su pplied from polycarbonate bottles attached to the cage and was available ad libitum. - Acclimation period: The animals were acclimatised for se ven days during which time their health status was assessed

.

ENVIRONMENTAL CONDITIONS - Temperature (°C): 22 ± 3 oC - Humidity (%): 50 ± 20 % - Air changes (per hr): The rate of air exchange was a t least fifteen air changes per hour. - Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hou rs continuous light and twelve hours darkness.

IN-LIFE DATES: From: To: 

## Administration / exposure

Route of administration

oral: gavage 

### Vehicle

arachis oil 

### Details on exposure

PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test item was prepared at the appropriat e concentrations as a solution in dried Arachis oil BP. The stability and homogeneity of the test item formulations were determined. Results show the formulations to be stable for at least eleven days. Bul k formulations were therefore generally prepared on a weekly basis and daily aliquot s stored at 4oC in the dark.

DIET PREPARATION - Rate of preparation of diet (frequency): No data - Mixing appropriate amounts with (Type of food): N ot applicable - Storage temperature of food: No data

VEHICLE - Justification for use and choice of vehicle (if other th an water): No data - Concentration in vehicle: 1.25, 5 and 15 mf/mL. - Amount of vehicle (if gavage): 4 mL/kg - Lot/batch no. (if required): No data - Purity: No data 

### Analytical verification of doses or concentrations

yes 

### Details on analytical verification of doses or conc entrations

METHOD OF ANALYSIS Summary The concentration of Benzenesulfonyl chloride in the te st item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Samples The test item formulations were extracted with acetonit rile to give a final, theoretical test item concentrat ion of approximately 0.02 mg/mL.

Standards Standard solutions of test item were prepared in aceto nitrile at a nominal concentration of 0.02 mg/ml.

Procedure The standard and sample solutions were analysed by HPLC using the following conditions: HPLC: Agilent Technologies 1200, incorporating autosa mpler and workstation Column: Sonoma C18 (150 x 4.6 mm id) Mobile phase: Acetonitrile: water (80:20 v/v) Flow-rate: 1 mL/min UV detector wavelength: 240 nm Injection volume: 25 μ L Retention time: ~ 3 mins Homogeneity Determinations The test item formulations were assessed visually. 

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2 of 13 26/07/2013 11:39Stability Determinations The test item formulations were sampled and analysed in itially and then after storage at approximately +4oC in the dark for eleven days.

Verification of Test Item Formulation Concentrations The test item formulations were sampled and analysed wi thin two days of preparation. 

### Details on mating procedure

- M/F ratio per cage: During the pairing phase, anima ls were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a on e male: one female basis within each dose group. - Length of cohabitation: No data - Proof of pregnancy: Vaginal plug - Further matings after two unsuccessful attempts: No dat a - After successful mating each pregnant female was caged ( how): Mated females were housed individually during g estation and lactation in solid floor polypropylene cages with stai nless steel mesh lids and softwood flakes. - Any other deviations from standard protocol: No data 

### Duration of treatment / exposure

The male dose groups were killed and examined macroscopica lly on Day 43. At Day 5 post partum, all females and surviving offspri ng were killed. 

### Frequency of treatment

Daily 

### Duration of test

The male dose groups were killed and examined macroscopica lly on Day 43. At Day 5 post partum, all females and surviving offspri ng were killed. 

### Doses / concentrations

5 mg/kg bw/day (equivalent to 1.25 mg/mL)

Basis actual ingested
20 mg/kg bw/day (equivalent to 5 mg/mL)

Basis actual ingested
60 mg/kg bw/day (equivalent to 15 mg/mL)

Basis actual ingested 

### No. of animals per sex per dose

12/sex/dose 

### Control animals

yes, concurrent vehicle 

### Further details on study design

- Dose selection rationale: - Rationale for animal assignment (if not random): The animals were allocated to dose groups using a random isation procedure based on stratified body weights, group mean body weights were then dete rmined to ensure similarity between the dose groups. 

## Examinations

Maternal examinations

Clinical Observations All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing during the working week. Animals were observed i mmediately before dosing soon after dosing and one hour after dosing at weekends (except for females during parturition where applicable). All observations were recorded.

Functional Observations Prior to the start of treatment and at weekly interva ls thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, after mating and prior to termination, together with an assessment o f sensory reactivity to various stimuli.

Behavioural Assessments Detailed individual clinical observations were performe d for each animal using a purpose built arena. The following parameters were observed: Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Ab normal/Stereotypic behaviour, Urination, Salivation, Defecation, Pilo-erection, Tra nsfer arousal, Exophthalmia, Tail elevation, Lachryma tion.

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioural Assessments and Sensory Reactivity Tests.

Functional Performance Tests Motor Activity. Purpose-built 44 infra-red beam automated activity mo nitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time ea ch day, under similar laboratory 

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3 of 13 26/07/2013 11:39conditions. The evaluation period was thirty minutes for each animal. The percentage of time each animal was active and mobile was recorded for the overall thirty minute period and also during the final 20% of the period (consider ed to be the asymptotic period, Reiter and Macphail, 1979).

Forelimb/Hindlimb Grip Strength. An automated meter was used. Each animal was allowed to grip the proximal metal bar of the meter with it s forepaws. The animal was pulled by the base of the tail until its grip was bro ken. The animal was drawn along the trough of the meter by the tail until its hind paws g ripped the distal metal bar. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecu tive trials were performed for each animal. The assessment was developed from the meth od employed by Meyer et al (1979).

Sensory Reactivity Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed fro m the methods employed by Irwin (1968) and Moser et al (1988). The following parameters were observed: Grasp response, Touch escape, Vocalisation, Pupil reflex, T oe pinch, Blink reflex, Tail pinch, Startle reflex, Fi nger approach.

Body Weight Individual body weights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until m ating was evident. Body weights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

Food Consumption During the pre-pairing period, weekly food consumption was recorded for each cage of adults. This was continued for males after the mating p hase. For females showing evidence of mating, food consumption was recorded for th e periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litte rs, food consumption was recorded on Days 1 and 4 post partum. Food efficiency (the ratio of body weight change/dieta ry intake) was calculated retrospectively for males throughout the study period (with the exception of the mating phase) and for fema les during the pre-pairing phase. Due to offspring growth and milk production, food efficiency could not be accurately calcula ted for females during gestation and lactation.

Water Consumption As an effect of water intake had been suspected for ani mals on the preliminary fourteen day toxicity study (Harlan Laboratiries Ltd. Project n umber 41201578), water consumption was measured daily throughout the study (wit h the exception of the pairing phase).

Reproduction Screening Mating Animals were paired on a 1 male: 1 female basis with in each dose group, for a period of up to fourteen days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined fo r the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of oestrus or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating (Day 0 of gestation) and the males were subsequently return ed to their original holding cages (unless required for additional pairing). Mated fema les were housed individually during the period of gestation and lactation.

Pregnancy and Parturition Each pregnant female was observed three times a day (at approximately 0830, 1230 and 1630 hours) during the normal working week and around the period of expected parturition. Observations were carried out twice daily (at approximately 0830 and 1230 hours) at weekends. Where possible the following wa s recorded for each female: i) Date of pairing ii) Date of mating iii) Date and time of observed start of parturition iv) Date and time of observed completion of parturiti on

Laboratory Investigations Haematological and blood chemical investigations were performed on five males and five females selected from each test and control group prior to termination (Day 42 for males and Day 4 post partum for females). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were taken by cardiac puncture at termination. Animals were not fasted prior to sampling.

Haematology The following parameters were measured on blood collect ed into tubes containing potassium EDTA anti-coagulant: Haemoglobin (Hb) Erythrocyte count (RBC) Haematocrit (Hct) Erythrocyte indices - mean corpuscular haemoglobin (MCH) - mean corpuscular volume (MCV) - mean corpuscular haemoglobin concentration (MCHC) Total leucocyte count (WBC) Differential leucocyte count - neutrophils (Neut) - lymphocytes (Lymph) - monocytes (Mono) 

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4 of 13 26/07/2013 11:39- eosinophils (Eos) - basophils (Bas) Platelet count (PLT) Reticulocyte count (Retic) - Methylene blue stained slide s were prepared but reticulocytes were not assessed Prothrombin time (CT) was assessed by ‘Innovin’ and Acti vated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collect ed into sodium citrate solution (0.11 mol/L).

Blood Chemistry The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant: Urea Calcium (Ca++) Glucose Inorganic phosphorus (P) Total protein (Tot.Prot.) Aspartate aminotransferase (ASAT) Albumin Alanine aminotransferase (ALAT) Albumin/Globulin (A/G) ratio (by calculation) Alkaline phosphatase (AP) Sodium (Na+) Creatinine (Creat) Potassium (K+) Total cholesterol (Chol) Chloride (Cl-) Total bilirubin (Bili) Bile acids

Pathology Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguinationon Day 5 post partum. Surviving offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any females which failed to achieve pregnancy were killed onor after Day 25 post coitum. For all females, the uterus was examined for signs of im plantation and the number of uterine implantations in each horn was recorded. This p rocedure was enhanced; as necessary, by staining the uteri with a 0.5% ammonium polysulphide solution (Salewski 1964). All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any m acroscopic abnormalities were recorded.

Organ Weights The following organs were dissected free from fat and weighed before fixation from five selected males and five selected females from each dose gro up: Adrenals, Prostate, Brain, Seminal vesicles, Epididymide s, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroi d (weighed post-fixation with Parathyroid), Ovaries, Uterus (weighed with Cervix), Pituitary (post fixation). Tissues below were weighed from all remaining animals : Prostate, seminal vesicles, epididymides, kidneys, testes, i terus (weighed with cervix) ovaries, pituitary (post fi xation).

Histopathology Samples of the following tissues were removed from fiv e selected males and five selected females from each dose group and preserved in b uffered 10 % formalin, except where stated: Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bon e marrow (femur including stifle joint), Pituitary, B one & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pon s), Oesophagus, Caecum, Rectum, Coagulating gland, Sal ivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epid idymides~, Skin (hind limb), Eyes*, Spinal cord (cervica l, mid-thoracic and lumbar), Gross lesions, Heart, Spleen, Ileum (including peyer’s patches) , Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trach ea, Liver, Testes , Lungs (with bronchi)#, Thymus, Lymph nodes (mandibular and mesenter ic), Urinary bladder, Mammary gland, Uterus/Cervix, M uscle (skeletal), Vagina

The following tissues were preserved from all remaining animals: Ovaries, pituitary, prostate, coagulating gland, semina l vesicles, epididymides, testes, uterau/cervix, vagina, m ammary gland. The tissues from five selected control and 1000 mg/kg bw/day dose group anim als, any animals dying during the study, and any animals which failed to achieve a pr egnancy were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μ m and stained with haematoxylin and eosin for subsequent microscopic examination. In add ition, sections of testes from all control and 60 mg/kg bw/day m ales were also stained with Periodic Acid-Schiff (PAS) stain and examined. Since there were indications of treatment-related chang es for the stomach of both sexes and the liver and kidneys of males at 60 mg/kg bw/day, examination was subsequently extended to include similarly prepared sections of stoma ch from five animals per sex from the low and intermediate groups and from five m ales from the low and intermediate groups for the liver and kidneys. * = eyes fixed in Davidson’s fluid = preserved in Bouin’s fluid then transferred to 70% Industrial Methylated Spirits (IMS) approximately forty-eight hours later # = lungs were inflated to approximately normal inspi ratory volume with buffered 10 % formalin before immersion in fixative 

### Fetal examinations

Litter Data On completion of parturition (Day 0 post partum), the number of live and dead offspring was recorded. Offspring were individually identified w ithin each litter by tattoo on Day 1 post partum. For each litter the following was recorded: i) Number of offspring born ii) Number of offspring alive recorded daily and repo rted on Days 1 and 4 post partum iii) Sex of offspring on Days 1 and 4 post partum iv) Clinical condition of offspring from birth to Day 5 post partum 

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5 of 13 26/07/2013 11:39v) Individual offspring weights on Days 1 and 4 post p artum (litter weights were calculated retrospectively from this data)

Physical Development All live offspring were assessed for surface righting refl ex on Day 1 post partum.

All offspring, including those dying during the study, were subjected to a full external and internal examination, and any m acroscopic abnormalities were recorded. 

### Statistics

Where considered appropriate, quantitative data was su bjected to statistical analysis to detect the significance of intergroup differences from c ontrol; statistical significance was achieved at a level of p<0.05. Statistical analysis wa s performed on the following parameters: Grip Strength, Motor Activity, Body Weight, Body Wei ght Change, Food Consumption during gestation and lactation, Water Consumption duri ng gestation and lactation, PreCoital Interval, Gestation Length, Litter Size, Litt er Weight, Sex Ratio, Corpora Lutea, Implantation Sites, Implantation Losses, Viability Indi ces, Offspring Body Weight, Offspring Body Weight Change, Offspring Surface Righti ng, Haematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relativ e Organ Weights. 

### Indices

Mating Performance and Fertility The following parameters were calculated from the indi vidual data during the mating period of the parenta l generation: i) Pre-coital Interval Calculated as the time elapsing between initial pairin g and the observation of positive evidence of mating.

ii) Fertility Indices For each group the following were calculated: Mating Index (%) = [Number of animals mated/Number of animals paired] x 100 Pregnancy Index (%) = [Number of pregnant females/ N umber of animals mated] x 100

Gestation and Parturition Data The following parameters were calculated for individua l data during the gestation and parturition period o f the parental generation. i) Gestation Length Calculated as the number of days of gestation including the day for observation of mating and the start of p arturition.

ii) Parturition Index The following was calculated for each group: Parturition Index (%) = [Number of females deliverin g live offspring/Number of pregnant females] x 100 

### Any other information on materials and methods incl . tables


## Litter Responses

The standard unit of assessment was considered to be the litter, therefore, values were first calculated for each litter and the group mean was calcu lated using the individual litter values. Group mean values included all litters reared to termi nation (Day 5 of age).

i) Implantation Losses (%) Group mean percentile pre-im plantation and post-implantation loss were calculated for each female/litter as follows:

Pre–implantation loss = [(Number of corpora lutea - nu mber of implantation sites)/ Number of corpora lutea] x100

Post–implantation loss = [(Number of implantation site s - Total number of offspring born)/Number of implantation sites]x100

ii) Live Birth and Viability Indices The following ind ices were calculated for each litter as follows:
Live Birth Index (%) = [Number of offspring alive o n Day 1/ Number of offspring born]x 100

Viability Index (%) = [Number of offspring alive on D ay 4/Number of offspring alive on Day 1] x 100
iii) Sex Ratio (% males) Sex ratio was calculated for each litter on Days 1 and 4 post partum, using the following formula:
[Number of male offspring/ Total number of offspring] x 100

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## RESULTS

Homogeneity of Test Item Formulations The test item formulations were deemed homogeneous by visual inspection

Stability of Test Item Formulations
Nominal concentration (mg/mL) Concentration found initially (mg/mL) Concentration found after storage for 11 days mg/mL Expressed as % of initial
1.25 1.22 1.24 102
250 253 251 99



## Verification of concentration of test item formulat ions

Analysis number Nominal concentration

(mg/mL) Concentration found

mg/mL Expressed as % of nominal

1 0 ND -

1.25 1.25 100 5 4.97 99 15 15.1 101 2 0 ND -

1.25 1.28 102 5 5.16 103 15 14.3 95 3 0 ND -

1.25 1.31 105 5 5.05 101 15 15.1 101 4 0 ND -

1.25 1.25 100 5 5.12 102 15 15.4 103 5 0 ND -

1.25 1.24 100 5 5.10 102 15 15.3 102 6 0 ND -

1.25 1.23 98 5 5.08 102 15 15.3 102

ND = none detected - = not applicable



## Conclusion The stability of the test item in the vehicle matri x during storage at approximately +4°C in the dark was confirmed to be at least eleven days. Dosing fo rmulations analysed during the study were within 98-105 % of nominal concentration and were consider ed to confirm the accuracy of the formulation procedure.

METHOD VALIDATION

Linearity A range of standard solutions covering the concentr ation range 0 to 0.0436 mg/ml, were prepared and analysed. The detector response was shown to be linear up to 0.0436 mg/ml.

Standard concentration (mg/mL) Peak Area (units)
0.000 0.000
0.0109 8.655E+-06 

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### 0.0164 1.313E+-07
0.0218 1.786E+-07
0.0327 2.651E+-07
0.0436 3.543E+-07 Slope 8.145E+-08 Intercept -9.253E+-04
Calculation 1.000



## Specificity The diluent solvent acetonitrile and a blank Dried Arachis Oil BP (control) were analysed. The results are shown in the following table:

Sample Concentration found
Acetonitrile None detected Dried arachis oil BP (control) None detected



## Analysis of the solvent and a blank Dried Arachis O il BP (control) produced no signal that interfered with the signal due to the test item.

Accuracy Samples of Dried Arachis Oil BP were accurately for tified with known amounts of test item, and analysed:

Fortification (mg/g) Concentration found

(mg/g) % recovered Mean recovery (%)
1.25 1.41 112 112
1.25 1.40 111
4.44 4.57 103 104
4.64 4.87 105
258 257 100 100
255 257 101



## The analytical method has been considered to be suf ficiently accurate for the purpose of this study. The test sample results have not been corrected forrecovery.

Conclusion The analytical method has been satisfactorily valid ated in terms of linearity, specificity and accurac y for the purposes of the study. Statistical analysis continued: 

## Where possible, data were analysed using the decision t ree from the ProvantisTM Tables and Statistics Module as detailed below: Where appropriate , data transformations were performed using the most suitable method. The homogeneity of var iance from mean values was analysed using Bartlett’s test. Intergroup variances wereassessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any t ransformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data w ere analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test t o determine significant difference from the control group. Where the data were unsuitable forthese analyses, pair-wise tests was performed using the Student t-test (parametric) or t he Mann-Whitney U test (nonparametric). Data not analysed by the Provantis data capture system wer e assessed separately using the SPSS statistical package. Initially, the homogeneity of the data was assessed using Levene’s test. Where Levene’s test was shown to be non-significant (p ≥ 0.05), parametric analysis of the data was applied, incorporating analysis of varianc e (ANOVA). If this data was shown to be significant, this analysis was followed by pair-wise comp arisons using Dunnett’s test. Where Levene’s test was significant, non-parametric an alysis of the data was analysed incorporating the Kruskal-Wallis test which if significan t, was followed by the Mann-Whitney U test. Dose response relationship was also be investigated b y linear regression. Where the data was unsuitable for these analyses, then pair-wise te sts were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric). Endpoint study record: Developmental toxicity / ter atogenicity. OECD 4... file:///C:/Documents and Settings/kwilloughby/Local Settings/Temp/ind...

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## Due to the preponderance of non-normally distributed data, reproductive parameters

(implantation losses, offspring sex ratio and offspring surface righting) were generally analysed using non-parametric analyses. Probability values (p) were calculated as follows: p<0.001 *** p<0.01** p<0.05* P ≥0.05 (not singificant) 

## In addition, suspected treatment-related histopathology findings were statistically analysed (excluding any decedents, non-mated females and females not producing a pregnancy/litter). 

# Results and discussions

Effect levels

Endpoint Effect type Effect level Based

on Basis for effect level / Remarks

NOEL developmental

toxicity 60 mg/kg bw/day

(actual dose received) test

mat. Evaluation of reproduction during the study did not indicate any obvious effect of

treatment and the No Observed Effect Level (NOEL) fo r reproduction including litter size and offspring survival, growth and development was 60 mg/kg bw/day. 

### Maternal toxic effects

yes (See results) 

### Details on maternal toxic effects

Mortality There were no deaths observed on the study that were considered to be related to treatment. At 60 mg/kg bw/ day, two males were terminated prior to scheduled termination due to suspected intubation error during the dosing procedure, both deaths were consi dered to be unrelated to treatment. Animal 77 was observed to have gasping respiration, le thargy, hunched posture and staining around the snout o n Day 23 and was killed for animal welfare considerations. Necropsy revealed a mass filled wi th clear fluid-like material under the left forelimb and dark lungs. Animal 78 was observed to have tip-toed gait, labored and decreased respiration rate, ptosis, pilo-erection, lethargy, hun ched posture and pallor of the extremities on Day 41 and was also killed for animal we lfare considerations. Necropsy revealed fibrous adhesions to the heart and lungs, gaseous distension of the stomach, duodenum, jejunum, ileum, ca ecum and colon, enlarges adrenals and dark liver, thymu s and lungs.

Clinical Observations The clinical signs observed on the study for surviving an imals were unremarkable and did not indicate any adverse effect of treatment at 5, 20 or 60 mg/kg bw/day.

Functional Observations Behavioural Assessments Assessment of the animals in a standard arena did not rev eal any obvious effects of treatment at 5, 20 or 60 mg /kg bw/day. The adverse clinical signs observed at 60 mg/kg bw/day during assessment on Day 41 were associated with animal 78. As previously discussed this animals was terminated early for animal welfare considerations and the underlying clause for the decline of this animal was considered to be intubation error, unrelated to treatment.

Functional Performance Tests Assessment of grip strength and motor activity did not i ndicate any obvious effect of treatment at 5, 20 or 60 mg/kg bw/day. Sensory Reactivity Assessments Sensory reactivity to different stimuli (auditory, visu al and proprioceptive) any obvious effect of treatment at 5, 20 or 60 mg/kg bw/day.

Body Weight There was no effect of treatment on body weight gain of males throughout the study, and body weight gain of treated males was frequently slightly superior to their control counterparts. Similarly body weight gain of females, including the periods of gesta tion and lactation, were not obviously affected by treatment during the study at 5, 20 or 60 mg/kg bw/day.

Food Consumption Food consumption for males throughout the pre and post pairing phases of the study and for females throughout the pre-pairing, gestation and lactation phases of the study was considered to have been unaffected by treatment. Food conversion efficiency for both sexes during pre-pairing phase and for males during the post-pairing phase was un affected by treatment at 5, 20 or 60 mg/kg bw/day.

Water Consumption At 20 and 60 mg/kg bw/day, water consumption for male s tended to be higher than control however there was no consistent dosage relationship and these differences probably represented normal biol ogical variation. There was no obvious effect of treatm ent on water intake for both sexes at 5 mg/kg bw/day or for females at 20 or 60 mg/kg bw/day

. Reproductive Performance Mating There were no effects of treatment on mating perform ance at 5, 20 or 60 mg/kg bw/day.

Fertility There were no obvious effects of treatment on fertili ty at 5, 20 or 60 mg/kg bw/day.

Gestation Length There were no effects of treatment on fertility at 5 , 20 or 60 mg/kg bw/day.

Laboratory Investigations Haematology For males at 60 mg/kg bw/day, mean blood haemoglobin, erythrocyte count and haematocrit were statistically sig nificantly lower than control; however for these treated animals only one value for erythrocyte count and two values for haematocrit were outside the historical control range. For males at 5 mg/kg bw/day lower mean platelet counts att ained statistical significance when compared to control. A s mean values showed no dosage relationship and all individual values were w ithin the historical control range, this finding was con sidered incidental and unrelated to treatment. There were no other statistically significant differences from control observed for haematology pa rameters for either sex at 5, 20 or 60 mg/kg bw/day. 

## Endpoint study record: Developmental toxicity / ter atogenicity. OECD 4... file:///C:/Documents and Settings/kwilloughby/Local Settings/Temp/ind...

9 of 13 26/07/2013 11:39Blood Chemistry For females at 60 mg/kg bw/day, higher cholesterol leve ls attained statistical significance when compared to cont rol; while four of the five values for treated animals exceeded the historical control range so did the same number of control values. There were no o ther statistically significant differences from control observed for blood chemistry parameters for either sex at 5, 20 or 60 mg/kg bw/day.

Pathology Necropsy - Offspring No treatment-related macroscopic abnormalities were d etected for interim death or terminal kill offspring. The incidental findings for offspring were typical for the age observed and were considered to be unrelated to toxicity of the test item.

Necropsy - Adults For surviving animals at 60 mg/kg bw/day, both sexes s howed an increased incidence of thickening of the non-gla ndular region of the stomach, compared to control, although only a minority of the animals was affected. Additionally for males at this do sage there was an increased incidence of enlargement and/or pallor of the kidneys. The in cidence of findings for either sex at 5 and 20 mg/kg bw /day did not indicate any obvious effect of treatment.

Organ Weights For males at 20 and 60 mg/kg bw/day there was an increa se in mean absolute and body weight-relative kidney weights compared with control, with differences attaining statistical significance. Th e majority of absolute and body weight relative valu es at these dosages exceeded the historical control range while only two control valuesfor absolute kidney weights exceeded this historical ra nge. Additionally for males at 20 mg/kg bw/day lower absolute and body weight-relative thyro id/parathyroid weights also attained statistical signifi cance when compared to control. However there was no similar decrease in thyroid/parathyroid weights for males at 60 mg/kg bw/day and in the absence of any histopathological findings at this high dosage level, the increase in thyroid/parathyroid was considered incidental and unrelat ed to treatment. For males at 20 mg/kg bw/day and females at all dosages, lower pituitary wei ght attained statistical significance when compared to control. However there was no dosage relationship and in the absence of any histopat hological findings for either sex at 60 mg/kg bw/day, the lower pituitary weights observed were considered incidental and unrelated to treatment

. Histopathology For males at 5, 20 and 60 mg/kg bw/day, examination o f the kidneys revealed a dosage related increase in the incidence and severity of foci of basophilic (regenerating) tubules, minimal to modera te granular cast formation and minimal or mild single cell death. Additionally for males at 60 mg/kg bw/day, minimal or mild centrilobular hypertrop hy of hepatocytes was observed in the liver. For both se xes at 60 mg/kg bw/day, minimal to moderate spongiosis (epithelial vacuolation), minimal t o moderate focal or diffuse epithelial hyperplasia of the squamous epithelium, minimal to moderate submucosal inflammation and, in one male, mi nimal hyperkeratosis in the non-glandular stomach was ob served. All of the other histopathological findings encountered were considered to have arisen spontaneously or at post mortem. 

### Embryotoxic / teratogenic effects

no effects 

### Details on embryotoxic / teratogenic effects

Litter Responses One female at 60 mg/kg bw/day showed total litter lo ss post partum and one control female was observed to ha ve a single implantation at necropsy but was not observed to have given birth to a litter. The following assessment is generally based on t he 10, 11, 10 and 9 litters successfully reared to Day 5 of age for the control, 5, 2 0 and 60 mg/kg bw/day dosage groups respectively.

Offspring Litter Size, Sex Ratio and Viability There was no adverse effect of maternal treatment on c orpora lutea and implantation counts, post-natal litt er size, offspring survival or sex ratio at 5, 20 or 60 mg/kg bw/day. At 60 mg/kg bw/day, female number 85 showed total litter loss post partum, however in the absence of any increased post-natal losses for other females at this dosage, compar ed to control, this finding was considered incidental a nd unrelated to treatment.

Offspring Growth and Development At 5, 20 or 60 mg/kg bw/day there was no effect of mat ernal treatment on initial offspring body weight or subsequent body weight gain to Day 4 of age. At 60 mg/kg bw/day, performance of offspring at D ay 1 of age during the assessment of surface righting ab ility was slightly inferior to control. However, differences from control did not attain stat istical significance and, in isolation, this finding was considered incidental and of no toxicological significance. At 5 and 20 mg/kg bw/day, per formance of the offspring at Day 1 of age was similar to control. Clinical signs observed for the offspring at at 5, 20 or 60 mg/kg bw/day wer e few and typical for the age observed and did not i ndicate any effect of maternal treatment on offspring development. 

# Overall remarks, attachments
Remarks on results including tables and figures



## Discussion

The oral administration of Benzenesulfonyl Chloride to rats for approximately six weeks (including two weeks pre-pairing, gestation and early lactatio n period for females) at dose levels of up to 60 mg/kg bw/day resulted in treatment related findings in animals of either sex receiving 60 mg/kg bw/day and in males receiving 5 or 20 mg/kg bw/day. Two animals were killed in extremis at 60 mg/kg bw/day but the decline in clinical condition for bo th animals was considered to be related to intubati on error during the dosing procedure and, as such, the se deaths were unrelated to treatment. Clinical signs for surviving animals were unremarkable and d id not give any indication of toxicity. There were considered to be no effects of treatment on behavio ural or functional assessments, body weight, food consumption or food conversion efficiency for either sex throughout the study. Water consumption at 20 and 60 mg/kg bw/day tended to be higher for males but showed no consistent dosage relationship; at the level observed, this wa s considered likely to reflect normal biological variation and was insufficient to represent any adv erse effect of treatment.

For males at 60 mg/kg bw/day, mean blood haemoglobi n, erythrocyte count and haematocrit were statistically significant lower than control. Howev er the majority of individual values for these trea ted males were within the historical control range and, in the absence of any histopathological correlates

, Endpoint study record: Developmental toxicity / ter atogenicity. OECD 4... file:///C:/Documents and Settings/kwilloughby/Local Settings/Temp/ind...

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## these differences were considered not to represent an adverse effect of treatment. For females at

60 mg/kg bw/day, higher cholesterol levels attained statistical significance when compared to control. While this finding may reflected altered liver meta bolism, as indicated microscopically by minimal/mil d centrilobular hypertrophy of the hepatocytes, in is olation, it was not to represent an adverse effect of treatment.

At 60 mg/kg bw/day, both sexes showed an increased incidence of thickening of the nonglandular region of the stomach at termination, compared to c ontrol, although only a minority of the animals was affected. Microscopic examination of the stomac h of both sexes, revealed minimal to moderate spongiosis (epithelial vacuolation), minimal to mod erate focal or diffuse epithelial hyperplasia of th e squamous epithelium, minimal to moderate submucosalinflammation and, in one male, minimal hyperkeratosis in the nonglandular stomach was obse rved. While these finding probably represent an adverse effect of treatment they are considered to reflect local irritation rather than any adverse systemic toxicity of the test item.

For males at 60 mg/kg bw/day there was also an incr eased incidence of enlargement and/or pallor of the kidneys observed at termination, and a statisti cally significant increase in absolute and body weight-relative kidney weights, compared with contr ol, was apparent for males at 20 and 60 mg/kg bw/day. Microscopic evaluation of the kidneys revea led a dosage-related increase in the incidence and severity of foci of basophilic (regenerating) t ubules, minimal to moderate granular cast formation and minimal or mild single cell death for males at 5, 20 and 60 mg/kg bw/day. The findings in the kidneys were considered to be consistent with a dia gnosis of alpha 2 μ macroglobulin nephropathy which is unique to the mature male rat and, as such , is of no relevance to humans. This finding is commonly observed in male rats following treatment with some hydrocarbons and is not predictive of any adverse effect in humans.

For males at 20 mg/kg bw/day statistically signific ant lower thyroid/parathyroid and pituitary weights were apparent, while for females at 5, 20 and 60 mg/kg bw/day, lower pituitary weights attained statistical significance compared to control. Howev er, in the absence of any dosage relationship or histopathological change at 60 mg/kg bw/day, these differences in organ weights were considered incidental and unrelated to treatment. For males at 60 mg/kg bw/day, microscopic examinati on of the liver revealed minimal or mild centrilobular hypertrophy of the hepatocytes, altho ugh this did not lead to any statistically signific ant differences in liver weights when compared with con trol. Hepatocyte enlargement is commonly observed in the rodent liver following the administ ration of xenobiotics and, in the absence of any associated inflammatory or degenerative changes, is generally considered to be adaptive in nature and does not represent an adverse health effect.

The microscopic changes observed for the male kidne y at all dosages and for the stomach in both sexes at 60 mg/kg bw/day make the allocation of a N o Observed Adverse Effect Level (NOAEL) for this study problematical. While the kidney changes observed for males clearly represent an adverse effect of treatment for the rat at dosages as low a s 5 mg/kg bw/day, the changes observed are not considered predictive of an effect in man. Excludin g these kidney changes, the NOAEL was 20 mg/kg bw/day due to adverse findings in the fore-st omach of both sexes at 60 mg/kg bw/day, although these changes probably represent local irr itation rather than a systemic toxic effect of the test item. Evaluation of reproduction during the st udy did not indicate any obvious effect of treatmen t and the No Observed Effect Level (NOEL) for reprodu ction including litter size and offspring survival, growth and development was 60 mg/kg bw/day. 

# Applicant's summary and conclusion

Conclusions

Microscopic kidney changes observed for male rats clearly r epresent an adverse effect of treatment for the rat a t dosages as low as 5 mg/kg bw/day, however the changes observed are not considered predictive of an effect in man. Excluding these kidney changes, the No Observed Adverse Effect Level (NOAEL) for all other findings wa s 20 mg/kg bw/day due to adverse microscopic fore-stomach findings for both sexes at 60 mg/kg bw/day, although these changes represent local irri tation rather than a systemic toxic effect of the test it em and therefore would not be included in subsequent assessment of risk. In terms of risk assessment, the findings observed on this study would sugge st that a NOAEL can be established at 60 mg/kg bw/day because the findings were either specific to male rats (kidney) or were not evid ence of systemic toxicity (stomach). Evaluation of reproduction during the stud y did not indicate any obvious effect of treatment an d the No Observed Effect Level (NOEL) for reproduction including litter size and offspring sur vival, growth and development was 60 mg/kg bw/day. 

### Executive summary



## Introduction.

The study was designed to investigate the systemic toxicity and potential adverse effects of the test item on reproduction (including offspring developme nt) and is compatible with the requirements of the Endpoint study record: Developmental toxicity / ter atogenicity. OECD 4... file:///C:/Documents and Settings/kwilloughby/Local Settings/Temp/ind...

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## OECD Guidelines for Testing of Chemicals No.

422 “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test” (adopted 22 March 1996). This study was also designed to be compatible with the Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Regis tration, Evaluation, Authorisation and Restriction of Chemicals (REACH). In addition it wa s also compatable with Toxic Substances Control Act (TSCA) "Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test" Code of Federal Regulations (CFR), Title 40, Protection of Environment, Pt. 799.9365.

Methods. The test item was administered by gavage to three g roups, each of twelve male and twelve female Wistar HanTM:RccHanTM:WIST strain rats, for approximate ly six weeks (including for females a two week pre-pairing phase, pairing, gestation and earl y lactation for females), at dose levels of 5, 20 and 60 mg/kg bw/day. A control group of twelve male s and twelve females was dosed with vehicle alone (dried Arachis oil BP) over the same treatmen t period. Clinical signs, behavioural assessments, body weigh t change, food and water consumption were monitored during the study. Pairing of animals with in each dose group was undertaken on a one male: one female basis within each treatment group on Day 15 of the study, with females subsequently being allowed to litter and rear theiroffspring to Day 5 of lactation. During the lactat ion phase, daily clinical observations were performed o n all surviving offspring, together with litter siz e and offspring weights and assessment of surface rig hting reflex. Extensive functional observations were performed onfive selected males from each dose group after the completion of the pairing phase, and for five selected parental females from each dose group on Day 4 post partum . Haematology and blood chemistry were evaluated pr ior to termination on five selected males and females from each dose g roup. Surviving adult males were terminated on Day 43, followed by the termination of all lactatin g females and offspring on Day 5 post partum . Any female which did not produce a pregnancy was termin ated on or after Day 25 post coitum . All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues was performed.

Results. Adult Responses: Mortality. There were no deaths observed on the study that wer e considered to be related to treatment. Clinical Observations. Clinical signs did not indicate any effect of treat ment at 5, 20 or 60 mg/kg bw/day. Behavioural Assessment. Assessment in a standard arena did not reveal any e ffects of treatment at 5, 20 or 60 mg/kg bw/day. Functional Performance Tests. Functional Performance Tests did not indicate any e ffect of treatment at 5, 20 or 60 mg/kg bw/day. Sensory Reactivity Assessments. Sensory reactivity did not indicate any effect of t reatment at 5, 20 or 60 mg/kg bw/day. Body Weight. Body weight and body weight gain did not indicate a ny effect of treatment at 5, 20 or 60 mg/kg bw/day. Food Consumption. Food consumption and food conversion efficiency didnot indicate any effect of treatment at 5, 20 or 60 mg/kg bw/day. Water Consumption. Water consumption was considered to have been unaff ected by treatment at 5, 20 or 60 mg/kg bw/day. Reproductive Performance: MatingMating performance was unaffected by treatment at 5 , 20 or 60 mg/kg bw/day. Fertility. Fertility was unaffected by treatment at 5, 20 or 6 0 mg/kg bw/day. Gestation Lengths. Gestation length was unaffected by treatment at 5, 20 or 60 mg/kg bw/day. Litter Responses: Offspring Litter Size, Sex Ratio and Viability. There were no effects of maternal treatment on numbers of corpora lutea or implantation counts, po st-natal litter size, offspring survival or sex rat io at 5, 20 or 60 mg/kg bw/day. Offspring Growth and Development. There was considered to be no effect of treatment o n offspring growth and development at 5, 20 or 60 mg/ kg bw/day. Laboratory Investigations: Haematology. For males at 60 mg/kg bw/day, mean blood haemoglobi n, erythrocyte count and Endpoint study record: Developmental toxicity / ter atogenicity. OECD 4... file:///C:/Documents and Settings/kwilloughby/Local Settings/Temp/ind...

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## haematocrit were statistically significantly lower than control.

Blood Chemistry. For females at 60 mg/kg bw/day, mean cholesterol le vels were statistically significantly higher than control. Pathology: Necropsy. For adults at 60 mg/kg bw/day, both sexes showed an increased incidence of thickening of the non-glandular region of the stomach, compare d to control. Additionally for males at 60 mg/kg bw/day, there was an increased incidence of enlarge ment and/or pallor of the kidneys. Necropsy of offspring did not indicate any effect of treatment at 5, 20 or 60 mg/kg bw/day. Organ Weights. For males at 20 and 60 mg/kg bw/day there was an st atistically significant increase in mean absolute and body weight-relative kidney we ights compared with control. Histopathology. For males at 5, 20 and 60 mg/kg bw/day, examination of the kidneys revealed a dosage-related increase in the incidence and severi ty of foci of basophilic (regenerating) tubules, minimal to moderate granular cast formation and min imal or mild single cell death. For males at 60 mg/kg bw/day, minimal or mild centrilobular hypertr ophy of hepatocytes was observed in the liver. For both sexes at 60 mg/kg bw/day, minimal to moderate spongiosis (epithelial vacuolation), minimal to moderate focal or diffuse epithelial hyperplasia of the squamous epithelium, minimal to moderate submucosal inflammation and, in one male, minimal h yperkeratosis in the non-glandular stomach was observed.

Conclusion. Microscopic kidney changes observed for male rats c learly represent an adverse effect of treatment for the rat at dosages as low as 5 mg/kg bw/day, ho wever the changes observed are not considered predictive of an effect in man. Excluding these kid ney changes, the No Observed Adverse Effect Level (NOAEL) for all other findings was 20 mg/kg bw/day due to adverse microscopic fore -stomach findings for both sexes at 60 mg/kg bw/day. These stomach changes represent local irrit ation rather than a systemic toxic effect of the test item and therefore would not be included in su bsequent assessment of risk. In terms of risk assessment, the findings observed on this study wou ld suggest that a NOAEL can be established at 60 mg/kg bw/day because the findings were either sp ecific to male rats (kidney) or were not evidence of systemic toxicity (stomach). Evaluation of reproduction during the study did notindicate any obvious effect of treatment and the No Observed Effect Level (NOEL) for reproduction in cluding litter size and offspring survival, growth and development was 60 mg/kg bw/day. Endpoint study record: Developmental toxicity / ter atogenicity. OECD 4... file:///C:/Documents and Settings/kwilloughby/Local Settings/Temp/ind...

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