Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

one-generation reproductive toxicity
based on generations indicated in Effect levels (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 July - 20 August 2012
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
according to
other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
GLP compliance:
Limit test:

Test material

Test material form:
solid: particulate/powder
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Sodium methallylsulphonate
- Substance type: White crystalline powder
- Physical state: Powder
- Purity: >99%
- Lot/batch No.: SD29052012/DD230312/LNR20120120
- Expiration date of the lot/batch: 06 June 2013
- Storage condition of test material: At room temperature in the dark
- Hygroscopic Yes, store in well-sealed container
- Volatile No
- pH: Expected to be ~neutral at concentration of 25%
- Stability in water: Expected to be stable for prolonged periods of time
- Solubility in water: >= 750 g/L

Test animals

other: Crl:WI(Han).
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: mean weight at start of treatment was 323 gr (males) or 211 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages.
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Temporary deviations from the daily mean level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 05 July - 20 August 2012

Administration / exposure

Route of administration:
oral: gavage
Details on exposure:
- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. No adjustment was made for specific gravity/density of the test substance, vehicle, and/or formulation. No correction was made for the purity/composition of the test substance.
- Storage conditions of formulations: At ambient temperature.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.
Details on mating procedure:
- M/F ratio per cage:1/1 (one female was cohabitated with one male of the same treatment group, avoiding sibling mating.
- Length of cohabitation: A maximum of 14 days was allowed for mating.
- Proof of pregnancy: Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated.
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type, height 18 cm).
Analytical verification of doses or concentrations:
Details on analytical verification of doses or concentrations:
The delegated phase was performed by the Principal Investigator for Formulation Analysis. Samples (0.5 mL) were taken using a pipette (a clean pipette tip was used for every group), and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 6 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice. Samples remained on dry ice until receipt at TNO Triskelion, The Netherlands, where samples were stored at ≤-18°C until analysis. Formulation samples were not transferred to the weighing room, but were dispatched directly to
the test site. Samples were received in good order by the test site.
Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%. No test substance was detected in the Group 1 formulations. The concentrations analysed in the formulations of Group 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours (i.e. relative difference ≤ 10%).
Duration of treatment / exposure:
Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-46 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One female of Group 1, one of group 2 and two females of group 3 were not dosed during littering.
Pups were not treated directly, but were potentially exposed to the test substance in utero and through lactational transfer.
Frequency of treatment:
Once daily for 7 d/w.
Details on study schedule:
- Age at mating: Approximately 13 weeks
Doses / concentrations
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/day
actual ingested
No. of animals per sex per dose:
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were based on a 90-day subchronic oral toxicity study (SC-94/0021). Wistar rats were treated at dose levels of 500, 2000 and 7000 ppm via drinking water corresponding to approximately 50, 250 and 1000 mg/kg. Adaptive responses were noted due to the high sodium uptake. These consisted of increased water consumption, renal epithelial cells in urine sediment and increase in urine protein. No toxicologically significant effects were demonstrated in animals treated with 7000 ppm, and therefore the NOAEL was considered to be 7000 ppm which corresponds to approximately 1000 mg/kg.
- Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (i.e. membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
- Identification of pups: On Day 1 of lactation, all pups were individually identified by means of subcutaneous injection of Indian ink.
Positive control:


Parental animals: Observations and examinations:
- Time schedule: At least twice daily.

- Time schedule: Daily, detailed clinical observations were made in all animals. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity.

- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

- (average food consumption [per animal per day]/average body weight per cage)x1000

Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.






- Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.
- Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth.
- Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.
Oestrous cyclicity (parental animals):
Not determined.
Sperm parameters (parental animals):
Slides of the testes were prepared for histopathological staging of spermatogenesis (all males of the control and high dose group and animals suspected to be infertile).
Litter observations:
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross abnomalies, weight gain, physical or behavioural abnormalities.

- Mortality: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
- Clinical signs: At least once daily, detailed clinical observations were made in all animals. No clinical observations were registered in the computer for 3 pups of one litter of Group 1 on Day 6 of lactation. Sufficient information available. Moreover, no findings were noted on the other days.
- Body weights: Live pups were weighed on Days 1 and 4 of lactation.
- Sex: Sex was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

Yes, if possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
- All animals were deeply anaesthetised and subsequently exsanguinated. The animals were not deprived of food overnight.
- According to test guidelines

- All animals: Epididymides, Kidneys, Liver, Testes
- The testes from one male of Group 4 was weighed after fixation. Fixation will not have a major impact on the testes weight.

- According to test guidelines
Postmortem examinations (offspring):
Pups surviving to planned termination were euthanised by subcutaneous injection of 0.03 mL pentobarbital (Euthasol® 20%; AST Farma B.V., Oudewater, The Netherlands) in the area between the scapulas on Days 5-7 of lactation.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. All pups were fixed in 10% buffered formalin. These were discarded after approval by the sponsor.

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 3 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Reproductive indices:
For each group, the following calculations were performed:
- Mating index: Number of females mated/Number of females paired x 100
- Fertility index: Number of pregnant females/Number of females paired x 100
- Conception index: Number of pregnant females/Number of females mated x 100
- Gestation index: Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index: Number of live pups on Day 4 of lactation / Number of pups born alive x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

No mortality occurred during the study period.

No clinical signs of toxicity were noted during the observation period. Salivation seen after dosing among five animals of the 1000 mg/kg dose group on 1 to 3 days during the treatment period was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. One female animal at 300 mg/kg showed alopecia and scabs on the right flank. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

Body weights and body weight gain of treated animals remained in the same range as controls over the treatment period.

Food consumption before or after allowance for body weight was similar between treated and control animals.

Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment. The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included diaphragmatic hernia of the liver, pelvic dilation of the kidneys, papillary process of the liver reduced in size, nodule on the epididymides or clitoral glands, focus on the clitoral glands, and alopecia.

No toxicologically relevant changes were noted in organ weights and organ to body weight ratios. The statistically significantly increased kidneys weights for males at 100 mg/kg were not considered toxicologically relevant as the increase was slight, no dose response was noted and all values were
within normal limits.

There were no treatment-related microscopic findings in the reproductive organs. No cause of infertility was found for the males that failed to sire (Group 1: 1, Group 4: 1) and the females that did not deliver healthy pups (Group 1: 1, Group 4: 1) Spermatogenic staging profiles were normal for all males examined. All microscopic findings recorded in animals surviving to the end of the assigned study period were within the normal range of background pathology encountered in Wistar Han rats of this age and strain.

No toxicologically relevant effects on reproductive parameters were noted. Mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

No toxicologically relevant effects on gestation index and duration, parturition and maternal care were observed. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth, and no deficiencies in maternal care were observed.

Effect levels (P0)

Dose descriptor:
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined

Details on results (F1)

No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

One pup of the control group was found dead on Day 2 of lactation and one pup at 300 mg/kg was missing on day 2 of lactation. The pup that was missing was most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

One pup at 100 mg/kg showed black discolouration of the tail apex and one pup at 1000 mg/kg showed red discolouration of the snout. The nature and incidence of these signs remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Effect levels (F1)

Dose descriptor:
Effect level:
> 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No toxicity was observed up to the highest dose level tested.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

In conclusion, treatment with Sodium methallylsulphonate by oral gavage in male and female Wistar Han rats at dose levels of 100, 300 and 1000 mg/kg revealed no parental, reproduction and developmental toxicity for treatment up to 1000 mg/kg. Based on these results, a parental, reproduction and developmental No Observed Adverse Effect Level (NOAEL) of at least 1000 mg/kg was derived.