Endpoint study record: Developmental toxicity / teratogenicity.

OECD 422 Benzenesulfonyl chloride LANXESS - 41201579

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 (formulation 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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Wistar

Details on test animals and environmental conditions

TEST ANIMALS

- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK.

- Age at study initiation: Approximately twelve weeks old.

- Weight at study initiation: At the start of treatment the males weighed 299 to 345 g, the females weighed

192 to 226 g.

- Fasting period before study: None

- Housing: Initially, all animals were housed in groups of four in solid floor polypropylene cages with

stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).

During the pairing phase, animals were transferred to polypropylene grid floor cages

suspended over trays lined with absorbent paper on a one 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 individually during gestation and

lactation in solid floor polypropylene cages with stainless steel mesh lids and softwood

flakes.

- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was available ad

libitum.

- Water (e.g. ad libitum): Mains drinking water was supplied from polycarbonate bottles attached to the cage and was available ad libitum.

- Acclimation period: The animals were acclimatised for seven 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 at least fifteen air changes per hour.

- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours 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 appropriate

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. Bulk formulations were therefore

generally prepared on a weekly basis and daily aliquots stored at 4oC in the dark.

DIET PREPARATION

- Rate of preparation of diet (frequency): No data

- Mixing appropriate amounts with (Type of food): Not applicable

- Storage temperature of food: No data

VEHICLE

- Justification for use and choice of vehicle (if other than 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 concentrations

METHOD OF ANALYSIS

Summary

The concentration of Benzenesulfonyl chloride in the test item formulations was determined by high performance liquid chromatography (HPLC)

using an external standard technique.

Samples

The test item formulations were extracted with acetonitrile to give a final, theoretical test item concentration of approximately 0.02 mg/mL.

Standards

Standard solutions of test item were prepared in acetonitrile 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 autosampler 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 μL

Retention time: ~ 3 mins

Homogeneity Determinations

The test item formulations were assessed visually.

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Stability Determinations

The test item formulations were sampled and analysed initially 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 within two days of preparation.

Details on mating procedure

- M/F ratio per cage: During the pairing phase, animals were transferred to polypropylene grid floor cages

suspended over trays lined with absorbent paper on a one 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 data

- After successful mating each pregnant female was caged (how): Mated females were housed individually during gestation and

lactation in solid floor polypropylene cages with stainless 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 macroscopically on Day 43.

At Day 5 post partum, all females and surviving offspring were killed.

Frequency of treatment

Daily

Duration of test

The male dose groups were killed and examined macroscopically on Day 43.

At Day 5 post partum, all females and surviving offspring 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 randomisation procedure based on stratified

body weights, group mean body weights were then determined 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 immediately 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 intervals 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 of sensory reactivity to various

stimuli.

Behavioural Assessments

Detailed individual clinical observations were performed 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/Abnormal/Stereotypic behaviour,

Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

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 monitors were used

to assess motor activity. Animals were randomly allocated to the activity monitors. The

tests were performed at approximately the same time each day, under similar laboratory

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conditions. 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 (considered 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 its forepaws. The animal was

pulled by the base of the tail until its grip was broken. The animal was drawn along the

trough of the meter by the tail until its hind paws gripped 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 consecutive trials were performed for

each animal. The assessment was developed from the method 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 from the methods employed by

Irwin (1968) and Moser et al (1988).

The following parameters were observed:

Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex, Finger 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 mating 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 phase. For females showing

evidence of mating, food consumption was recorded for the periods covering post coitum

Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded

on Days 1 and 4 post partum.

Food efficiency (the ratio of body weight change/dietary intake) was calculated

retrospectively for males throughout the study period (with the exception of the mating phase) and for females during the pre-pairing phase. Due to

offspring growth and milk

production, food efficiency could not be accurately calculated for females during

gestation and lactation.

Water Consumption

As an effect of water intake had been suspected for animals on the preliminary fourteen

day toxicity study (Harlan Laboratiries Ltd. Project number 41201578), water

consumption was measured daily throughout the study (with the exception of the pairing

phase).

Reproduction Screening

Mating

Animals were paired on a 1 male: 1 female basis within 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 for 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 returned to their original holding cages

(unless required for additional pairing). Mated females 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 was 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 parturition

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 collected 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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- eosinophils (Eos)

- basophils (Bas)

Platelet count (PLT)

Reticulocyte count (Retic) - Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin

time (APTT) was assessed by ‘Actin FS’ using samples collected 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 exsanguination on Day 5 post partum. Surviving

offspring were terminated via intracardiac overdose of sodium pentobarbitone. Any

females which failed to achieve pregnancy were killed on or after Day 25 post coitum.

For all females, the uterus was examined for signs of implantation and the number of

uterine implantations in each horn was recorded. This procedure 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 macroscopic 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 group:

Adrenals, Prostate, Brain, Seminal vesicles, Epididymides, Spleen, Heart, Testes, Kidneys, Thymus, Liver, Thyroid (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, iterus (weighed with cervix) ovaries, pituitary (post fixation).

Histopathology

Samples of the following tissues were removed from five selected males and five

selected females from each dose group and preserved in buffered 10 % formalin, except

where stated:

Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum),

Prostate, Brain (including cerebrum, cerebellum and pons), Oesophagus, Caecum, Rectum, Coagulating gland, Salivary glands (submaxillary),

Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides~, Skin (hind limb), Eyes*, Spinal cord (cervical, mid-thoracic and lumbar), Gross

lesions, Heart, Spleen, Ileum (including peyer’s patches), Stomach, Jejunum, Thyroid/parathyroid, Kidneys, Trachea, Liver, Testes , Lungs (with

bronchi)#, Thymus, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary gland, Uterus/Cervix, Muscle (skeletal), Vagina

The following tissues were preserved from all remaining animals:

Ovaries, pituitary, prostate, coagulating gland, seminal vesicles, epididymides, testes, uterau/cervix, vagina, mammary gland.

The tissues from five

selected control and 1000 mg/kg bw/day dose group animals, any animals dying during

the study, and any animals which failed to achieve a pregnancy were prepared as

paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with haematoxylin

and eosin for subsequent microscopic examination. In addition,

sections of testes from all control and 60 mg/kg bw/day males were also stained with

Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related changes 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 stomach from five animals per sex

from the low and intermediate groups and from five males 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 inspiratory 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 within 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 reported 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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v) Individual offspring weights on Days 1 and 4 post partum (litter weights were

calculated retrospectively from this data)

Physical Development

All live offspring were assessed for surface righting reflex on Day 1 post partum.

All offspring, including those dying during the study, were subjected to

a full external and internal examination, and any macroscopic abnormalities were

recorded.

Statistics

Where considered appropriate, quantitative data was subjected to statistical analysis to

detect the significance of intergroup differences from control; statistical significance was

achieved at a level of p<0.05. Statistical analysis was performed on the following

parameters:

Grip Strength, Motor Activity, Body Weight, Body Weight Change, Food Consumption

during gestation and lactation, Water Consumption during gestation and lactation, PreCoital Interval, Gestation Length, Litter Size, Litter Weight, Sex Ratio, Corpora Lutea,

Implantation Sites, Implantation Losses, Viability Indices, Offspring Body Weight,

Offspring Body Weight Change, Offspring Surface Righting, Haematology, Blood

Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.

Indices

Mating Performance and Fertility

The following parameters were calculated from the individual data during the mating period of the parental generation:

i) Pre-coital Interval

Calculated as the time elapsing between initial pairing 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/ Number of animals mated] x 100

Gestation and Parturition Data

The following parameters were calculated for individual data during the gestation and parturition period of 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 parturition.

ii) Parturition Index

The following was calculated for each group:

Parturition Index (%) = [Number of females delivering 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 calculated using the individual litter values.

Group mean values included all litters reared to termination (Day 5 of age).

i) Implantation Losses (%) Group mean percentile pre-implantation and post-implantation loss

were calculated for each female/litter as follows:

Pre–implantation loss = [(Number of corpora lutea - number of implantation sites)/ Number of

corpora lutea] x100

Post–implantation loss = [(Number of implantation sites - Total number of offspring

born)/Number of implantation sites]x100

ii) Live Birth and Viability Indices The following indices were calculated for each litter as

follows:

Live Birth Index (%) = [Number of offspring alive on Day 1/ Number of offspring born]x 100

Viability Index (%) = [Number of offspring alive on Day 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

Further information on analytical verification:

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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 formulations

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 matrix during storage at approximately +4°C in the dark

was confirmed to be at least eleven days. Dosing formulations analysed during the study were within

98-105 % of nominal concentration and were considered to confirm the accuracy of the formulation

procedure.

METHOD VALIDATION

Linearity

A range of standard solutions covering the concentration 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 Oil BP (control) produced no signal that interfered

with the signal due to the test item.

Accuracy

Samples of Dried Arachis Oil BP were accurately fortified 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 sufficiently accurate for the purpose of this study.

The test sample results have not been corrected for recovery.

Conclusion

The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy

for the purposes of the study.

Statistical analysis continued:

Where possible, data were analysed using the decision tree from the ProvantisTM Tables and

Statistics Module as detailed below: Where appropriate, data transformations were performed

using the most suitable method. The homogeneity of variance from mean values was

analysed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or

if required, ANCOVA with appropriate covariates. Any transformed 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 were analysed by a stepwise

Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from

the control group. Where the data were unsuitable for these analyses, pair-wise tests was

performed using the Student t-test (parametric) or the Mann-Whitney U test (nonparametric).

Data not analysed by the Provantis data capture system were 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 variance (ANOVA). If this data was shown to

be significant, this analysis was followed by pair-wise comparisons using Dunnett’s test.

Where Levene’s test was significant, non-parametric analysis of the data was analysed

incorporating the Kruskal-Wallis test which if significant, was followed by the Mann-Whitney U

test. Dose response relationship was also be investigated by linear regression. Where the

data was unsuitable for these analyses, then pair-wise tests were performed using the

Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

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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) for 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 considered to be unrelated to treatment.

Animal 77 was observed to have gasping respiration, lethargy, hunched posture and staining around the snout on Day 23 and was killed for animal

welfare considerations. Necropsy revealed a mass filled with 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, hunched posture and pallor of the

extremities on Day 41 and was also killed for animal welfare considerations. Necropsy revealed fibrous adhesions to the heart and lungs, gaseous

distension of the stomach, duodenum, jejunum, ileum, caecum and colon, enlarges adrenals and dark liver, thymus and lungs.

Clinical Observations

The clinical signs observed on the study for surviving animals 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 reveal 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 indicate any obvious effect of treatment at 5, 20 or 60 mg/kg bw/day.

Sensory Reactivity Assessments

Sensory reactivity to different stimuli (auditory, visual 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 gestation 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 unaffected by treatment at 5, 20 or 60 mg/kg bw/day.

Water Consumption

At 20 and 60 mg/kg bw/day, water consumption for males tended to be higher than control however there was no consistent dosage relationship

and these differences probably represented normal biological variation. There was no obvious effect of treatment 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 performance at 5, 20 or 60 mg/kg bw/day.

Fertility

There were no obvious effects of treatment on fertility 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 significantly 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 attained statistical significance when compared to control. As mean values showed no

dosage relationship and all individual values were within the historical control range, this finding was considered incidental and unrelated to

treatment. There were no other statistically significant differences from control observed for haematology parameters for either sex at 5, 20 or 60

mg/kg bw/day.

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Blood Chemistry

For females at 60 mg/kg bw/day, higher cholesterol levels attained statistical significance when compared to control; 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 other 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 detected 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 showed an increased incidence of thickening of the non-glandular region of the stomach,

compared to control, although only a minority of the animals was affected. Additionally for males at this dosage there was an increased incidence

of enlargement and/or pallor of the kidneys. The incidence 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 increase in mean absolute and body weight-relative kidney weights compared with control,

with differences attaining statistical significance. The majority of absolute and body weight relative values at these dosages exceeded the

historical control range while only two control values for absolute kidney weights exceeded this historical range. Additionally for males at 20 mg/kg

bw/day lower absolute and body weight-relative thyroid/parathyroid weights also attained statistical significance 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 unrelated to treatment. For males at 20 mg/kg

bw/day and females at all dosages, lower pituitary weight attained statistical significance when compared to control. However there was no

dosage relationship and in the absence of any histopathological 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 of the kidneys revealed a dosage related increase in the incidence and severity of foci of

basophilic (regenerating) tubules, minimal to moderate granular cast formation and minimal or mild single cell death. Additionally for males at 60

mg/kg bw/day, minimal or mild centrilobular hypertrophy 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 hyperkeratosis in the non-glandular stomach was observed. 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 loss post partum and one control female was observed to have a single implantation at

necropsy but was not observed to have given birth to a litter. The following assessment is generally based on the 10, 11, 10 and 9 litters

successfully reared to Day 5 of age for the control, 5, 20 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 corpora lutea and implantation counts, post-natal litter 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, compared to control, this finding was considered incidental and unrelated to treatment.

Offspring Growth and Development

At 5, 20 or 60 mg/kg bw/day there was no effect of maternal 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 Day 1 of age during the assessment of surface righting ability was slightly inferior to control.

However, differences from control did not attain statistical significance and, in isolation, this finding was considered incidental and of no

toxicological significance. At 5 and 20 mg/kg bw/day, performance 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 were few and typical for the age observed and did not indicate 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 lactation 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 killedin extremisat 60 mg/kg

bw/day but the decline in clinical condition for both animals was considered to be related to intubation

error during the dosing procedure and, as such, these deaths were unrelated to treatment. Clinical

signs for surviving animals were unremarkable and did not give any indication of toxicity. There were

considered to be no effects of treatment on behavioural 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 was considered likely to reflect normal biological

variation and was insufficient to represent any adverse effect of treatment.

For males at 60 mg/kg bw/day, mean blood haemoglobin, erythrocyte count and haematocrit were

statistically significant lower than control. However the majority of individual values for these treated

males were within the historical control range and, in the absence of any histopathological correlates,

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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 metabolism, as indicated microscopically by minimal/mild

centrilobular hypertrophy of the hepatocytes, in isolation, 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 control, although only a minority of the animals

was affected. Microscopic examination of the stomach of both sexes, revealed 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

hyperkeratosis in the nonglandular stomach was observed. 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 increased incidence of enlargement and/or pallor of

the kidneys observed at termination, and a statistically significant increase in absolute and body

weight-relative kidney weights, compared with control, was apparent for males at 20 and 60 mg/kg

bw/day. Microscopic evaluation of the kidneys revealed a dosage-related increase in the incidence

and severity of foci of basophilic (regenerating) tubules, 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 diagnosis of alpha 2μ 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 significant 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. However, 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 examination of the liver revealed minimal or mild

centrilobular hypertrophy of the hepatocytes, although this did not lead to any statistically significant

differences in liver weights when compared with control. Hepatocyte enlargement is commonly

observed in the rodent liver following the administration 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 kidney at all dosages and for the stomach in both

sexes at 60 mg/kg bw/day make the allocation of a No 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 as 5 mg/kg bw/day, the changes observed are not

considered predictive of an effect in man. Excluding these kidney changes, the NOAEL was 20

mg/kg bw/day due to adverse findings in the fore-stomach of both sexes at 60 mg/kg bw/day,

although these changes probably represent local irritation rather than a systemic toxic effect of the

test item. Evaluation of reproduction during the study did not indicate any obvious effect of treatment

and the No Observed Effect Level (NOEL) for reproduction 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 represent an adverse effect of treatment for the rat at 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 was 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 irritation rather than a systemic toxic effect of the test item and therefore would not be

included in subsequent assessment of risk. In terms of risk assessment, the findings observed on this study would suggest 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 evidence of systemic toxicity

(stomach). Evaluation of reproduction during the study did not indicate any obvious effect of treatment and the No Observed Effect Level (NOEL)

for reproduction including litter size and offspring survival, 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 development) and is compatible with the requirements of the

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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 Registration, Evaluation, Authorisation and

Restriction of Chemicals (REACH). In addition it was 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 groups, each of twelve male and twelve female

Wistar HanTM:RccHanTM:WIST strain rats, for approximately six weeks (including for females a two

week pre-pairing phase, pairing, gestation and early lactation for females), at dose levels of 5, 20

and 60 mg/kg bw/day. A control group of twelve males and twelve females was dosed with vehicle

alone (dried Arachis oil BP) over the same treatment period.

Clinical signs, behavioural assessments, body weight change, food and water consumption were

monitored during the study. Pairing of animals within 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 their offspring to Day 5 of lactation. During the lactation

phase, daily clinical observations were performed on all surviving offspring, together with litter size

and offspring weights and assessment of surface righting reflex.

Extensive functional observations were performed on five 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 4post partum. Haematology and blood chemistry were evaluated prior to termination

on five selected males and females from each dose group. Surviving adult males were terminated on

Day 43, followed by the termination of all lactating females and offspring on Day 5post partum. Any

female which did not produce a pregnancy was terminated on or after Day 25post 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 were considered to be related to

treatment.

Clinical Observations.Clinical signs did not indicate any effect of treatment at 5, 20 or 60 mg/kg

bw/day.

Behavioural Assessment.Assessment in a standard arena did not reveal any effects of treatment

at 5, 20 or 60 mg/kg bw/day.

Functional Performance Tests.Functional Performance Tests did not indicate any effect of

treatment at 5, 20 or 60 mg/kg bw/day.

Sensory Reactivity Assessments.Sensory reactivity did not indicate any effect of treatment at 5,

20 or 60 mg/kg bw/day.

Body Weight.Body weight and body weight gain did not indicate any effect of treatment at 5, 20 or

60 mg/kg bw/day.

Food Consumption.Food consumption and food conversion efficiency did not indicate any effect of

treatment at 5, 20 or 60 mg/kg bw/day.

Water Consumption.Water consumption was considered to have been unaffected 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 60 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, post-natal litter size, offspring survival or sex ratio

at 5, 20 or 60 mg/kg bw/day.

Offspring Growth and Development.There was considered to be no effect of treatment on

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 haemoglobin, erythrocyte count and

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haematocrit were statistically significantly lower than control.

Blood Chemistry.For females at 60 mg/kg bw/day, mean cholesterol levels 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, compared to control. Additionally for males at 60 mg/kg

bw/day, there was an increased incidence of enlargement 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 statistically significant increase

in mean absolute and body weight-relative kidney weights 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 severity of foci of basophilic (regenerating) tubules,

minimal to moderate granular cast formation and minimal or mild single cell death. For males at 60

mg/kg bw/day, minimal or mild centrilobular hypertrophy 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 hyperkeratosis in the non-glandular stomach was

observed.

Conclusion.

Microscopic kidney changes observed for male rats clearly represent an adverse effect of treatment

for the rat at 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

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 irritation rather than a systemic toxic effect of the

test item and therefore would not be included in subsequent assessment of risk. In terms of risk

assessment, the findings observed on this study would suggest 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

evidence of systemic toxicity (stomach).

Evaluation of reproduction during the study did not indicate any obvious effect of treatment and the

No Observed Effect Level (NOEL) for reproduction including litter size and offspring survival, growth

and development was 60 mg/kg bw/day.

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