Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 2007 - June 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed according to the OECD Consensus document Number 13 The Application of the OECD Principles of GLP to the Organization and Management of Multi-Site studies. The study procedures described in this report were based on the following guidelines: 1) Organisation of Economic Co-operation and Development Guidelines (OECD) for testing of Chemicals Guideline 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996. 2) Environmental Protection Agency (EPA) guideline 40 CFR 799.9365, TSCA combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, December 2000.
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EPA guideline 40 CFR 799.9365
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
• Source: Charles River Deutschland, Sulzfeld, Germany.
• Age at study initiation: ca. 10 weeks
• Number of animals: 40 females and 40 males. This resulted in at least 8 pregnant females per group.
• Weight at study initiation: range 290 - 293 g (males), 188-193 g (females)
• Acclimatisation:  5 d
• Accommodation:
- Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm).
- Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages (MIII type, height 18 cm).
- Post-mating: Males were housed in their home cage (Macrolon cages, MIV type, height 18 cm) with a maximum of 5 animals/sex/cage. Females were individually housed in Macrolon cages (MIII type, height 18 cm).
• Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), except the night prior to planned necropsy
• Water: Free access to tap-water.
• Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
• Temperature (°C): 21 ± 3
• Relative humidity (%): 30 – 70
• Air changes (1/h): ca. 15
• Photoperiod (h dark / h light): 12/12 (hours artificial light/ hours darkness per day).

IN-LIFE DATES:
• From August 29 (delivery of animals)to October 26, 2007

Route of administration:
oral: gavage
Type of inhalation exposure (if applicable):
other: not applicable
Vehicle:
water
Details on exposure:
Treatment
Parental animals
• Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.
• Frequency: once daily for 7 d/w , approximately the same time each day with a maximum of 4 h difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
• Exposure period:
- Males were exposed for 30 d, i.e. 2 w prior to mating, during mating, and up to termination.
- Females were exposed for 42 - 53 d, i.e. 2 w prior to mating, during mating, during post-coitum, and during at least 4 d of lactation.
• Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Offspring
• Offspring was not treated.
Details on mating procedure:
- M/F ratio per cage:
5 animals/sex/cage (Pre-Mating)
1:1 (mating)
5 male animals/cage (post-mating); females were housed individually (post-mating)

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling and analysis of formulations prepared on one day during the treatment period was performed according to the following scheme:
Group Analysis (type of sample)
1 acc (M)
2 acc + hom + stab{t=o} (TMB), stab{t=5, RT} (S)
3 acc (M)
4 acc + hom + stab{t=o} (TMB), stab{t=5, RT} (S)
with: acc=accuracy, hom=homogeneity, stab=stability (hours), T=top, M=middle, B=bottom position of container, S=stability sample taken at middle position of container, RT=room temperature
The analytical method used was based on the results of a separate project for the development and validation of the analytical method (HPLC; NOTOX project 485209)

For each dose group reproduction parameters were expressed in two ways:
- As a mean (with standard deviation) of the number observed for each litter
- Relative to a second parameter and calculated on a total group basis

Analysis of Dose Preparations:
The pH of the formulations were between 8.31 to 8.68 (Group 1), 10.12 to 10,17 (Group 2), 10.10 to 10.17 (Group 3) and 9.94 to 10.02 (Group 4).
The accuracies of the formulation of Group 2 were between 102 % and 109 %. For Group 3, accuracies of 98 % and 103 % were obtained and for Group 4 the accuracies were between 87 % and 105 %, The value of 87 % in Group 4 was accepted since the accuracy of the duplicate sampie was within the criterion range of 90 - 110 %. Based on the results obtained it was concluded that the formulations were prepared correctly.
No test substance was detected in the Group 1 formulations.
The formulations of Group 2 and Group 4 were homogeneous (coefficient of variation <= 10%).
Formulations at the entire range were stable for at least 5 h when stored at room temperature.
Duration of treatment / exposure:
Four groups of ten male and ten female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day.
Males were exposed for 30 d, i.e. 2 w prior to mating, during mating, and up to termination. Females were exposed for 42 to 53 d (during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 d of lactation).
Frequency of treatment:
Once daily (7 d/w), approx. the same time each day with max. 4 h difference between the earliest and latest dose. Animals were dosed up to the day prior to scheduled necropsy.
Details on study schedule:
BREEDING PROCEDURES:
Following a minimum of 14 d of exposure for the males and females, one female was cohabitated with one male of the same treatment group, avoiding sibling mating (Charles River will supply non-litter mates). Detection of mating was confirmed by evidence of sperm in the vaginal lavage or by the appearance of an intra-vaginal copulatory plug. This day was designated day 0 post-coitum.
Once mating had occurred, the males and females were separated. A maximum of 14 d was allowed for mating.
Parturition: The females were allowed to litter normally. Day 1 of lactation was defined as the day when a litter was found completed (membranes, placentas cleaned up, nest built up and/or feeding of pups started). Females that were littering were left undisturbed.
Remarks:
Doses / Concentrations:
5 mL/kg body weight
Basis:
other: Dose Volume
Remarks:
Doses / Concentrations:
0 mg/kg b.w.
Basis:
nominal in water
control group
Remarks:
Doses / Concentrations:
100 mg/kg b.w.
Basis:
nominal in water
10 females/10 males
Remarks:
Doses / Concentrations:
300 mg/kg b.w.
Basis:
nominal in water
10 females/10 males
Remarks:
Doses / Concentrations:
1,000 mg/kg b.w.
Basis:
nominal in water
10 females/10 males
No. of animals per sex per dose:
10 males: 0/100/300/1000 mg/kg b.w.
10 females: 0/100/300/1000 mg/kg b.w.
Offspring was not treated.
Control animals:
yes, concurrent no treatment
Details on study design:
• Dose selection rationale: range-finding study. Two dose levels (500 and 1,000 mg/kg bw ) were used to set the dose level for the main study. No mortality, clinical signs, macroscopic findings or organ weight effects were observed at these dose levels. A very slight effect on body weight and food consumption was observed for both groups which recovered at the end.
• Rationale for animal assignment (if not random): randomized
• Rationale for selecting satellite groups: not applicable
• Post-exposure recovery period in satellite groups: not applicable
• Section schedule rationale (if not random): not applicable
Positive control:
No positive control used in the study.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes (parental animals + offspring)
Parental animals:
Mortality: at least twice daily; Clinical signs: at least once daily
The following tests were performed in the first five mated males and females/group with live offspring:
hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent) motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England). During the motor activity test, males were caged individually and females were caged with their offspring. The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling). In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 min.
Offspring:
Mortality: the numbers of live and dead pups at the First Litter Check (= check at day 1 of lactation) and daily thereafter was determined.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: once daily
Body weight: Yes (parental animals + offspring)
Parental animals: Males and females were weighed on the first day of exposure and weekly there-after. Mated females were weighed on days 0, 4, 7, 11, 14, 17 and 20 of gestation, and during lactation on days 1 and 4.
Offspring: Live pups were weighed during lactation on days 1 and 4

FOOD CONSUMPTION AND COMPOUND INTAKE:
Weekly, for males and females. Food consumption was not recorded during the breeding period. Food consumption of mated females was measured on days 0, 4, 7, 11, 14, 17 and 20 postcoitum and after delivery on days 1 and 4 post-partum.

REPRODUCTION PROCESSES:
Male number paired with, mating date, confirmation of pregnancy, and delivery day was recorded.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
HAEMATOLOGY: Yes
Time schedule for collection of blood: prior to scheduled postmortem examination, between 7.00 and 10.30 a.m.
Anesthetic used for blood collection: Yes (iso-flurane)
Animals fasted: Yes overnight (with a maximum of 20 hours)
Animal number: the first five mated males/group and the first five females/group

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: prior to scheduled postmortem examination, between 7.00 and 10.30 a.m.
Animals fasted: Yes overnight (with a maximum of 20 hours)
Animal number: the first five mated males/group and the first five females/group

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Time schedule for examinations: after delivery of offspring
Dose groups that were examined: the first five mated males/group and the first five females/group with live offspring
attery of functions tested: hearing ability, pupillary reflex, static righting reflex and grip strength (Score 0 = normal/present, score 1 = abnormal/absent) motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England). During the motor activity test, males were caged individually and females were caged with their offspring. The assigned males were tested during week 4 of treatment and the assigned females were tested during lactation (all before blood sampling). In order to avoid hypothermia of pups, dams were removed from the pups for not more than 30-40 min.



Oestrous cyclicity (parental animals):
Not applicable
Sperm parameters (parental animals):
Parameters examined in male parental generations: No sperm parameters were examined.
see also "Parental animals: Observations and examinations"
Litter observations:
The following parameters were examined in offspring:
Mortality/Viability: The numbers of live and dead pups at the First Litter Check (= check at Day 1 of lactation) and daily thereafter was determined.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed during lactation on Days 1 and 4.
Sex: Was determined for all pups on Days 1 and 4 of lactation (by assessment of the ano-genital distance).

GROSS EXAMINATION OF DEAD PUPS:
All offspring was sexed and externally examined. The stomach was examined for the presence of milk. Descriptions of all external abnormalities were recorded. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
• Male animals: Following completion of the mating period (a minimum of 28 d of dose administration), the males were anaesthetised using iso-flurane (Abbott Laboratories, Zwolle, The Netherlands) and subsequently exsanguinated, Organ weights were collected and tissues were preserved for possible future histopathological examination as described in the following paragraphs.

• Maternal animals:
- Females which deliver: On lactation Day 5 or shortly thereafter, the females were anaesthetised using iso-flurane (Abbott Laboratories, Zwolle, The Netherlands) and subsequently exsanguinated.
- Females which failed to deliver: On post-mating day 24-26, the females which had not delivered were anaesthetised using iso-flurane (Abbott Laboratories, Zwolle, The Netherlands) and subsequently exsanguinated.

GROSS NECROPSY:
All animals were fasted overnight (with a maximum of 20 h) prior to planned necropsy, but water was provided.
• Females which deliver: Organ weights were collected and tissues were preserved for possible future histopathological examination as described in the following paragraphs. The number of former implantation sites was counted and recorded. Corpora lutea was also counted and recorded. Gross lesions were saved for possible future histopathological examination.
• Females which failed to deliver:
No organ weights were collected. Tissues were preserved for possible future histopathological examination as described in the following paragraphs, with the following exceptions: Nongravid uteri were stained using the Salewski technique (Salewski, 1964) in order to determine any former implantation sites (Salewski staining prepared at NOTOX using ammonium sulfide solution 20% (Merck, Darmstadt, Germany) and Milli-Ro water (Millipore Corporation, Bedford, USA)). Gross lesions were saved for possible future histopathological examination.

HISTOPATHOLOGY / ORGAN WEIGHTS:
Organ weights: YES
The following organ weights (and terminal body weight) were recorded:
From 5 surviving animals/sex/group (The first 5 mated males per group and the first 5 females with live offspring per group): Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus.
From all remaining males: Epididymides, Testes.
The tissues were prepared for microscopic examination and weighed, respectively.
Statistics:
The following statistical methods were used to analyse the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (Dunnett, 1955) (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 (Miller, 1981) (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution. The Fisher Exact-test (Fisher, 1950) 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. No statistical analysis was performed on histopathology findings. 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.
Reproductive indices:
For each dose group reproduction parameters were expressed in two ways:
- As a mean (with standard deviation) of the number observed for each litter
- Relative to a second parameter and calculated on a total group basis
Offspring viability indices:
The Viability index was calculated as follows:
[number of live pups on day 4 post partum/number of pups born alive]*100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see "details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
see "details on results"
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see "details on results"
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: see "details on results"
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
see "details on results"
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
see "details on results"
Reproductive performance:
no effects observed
Description (incidence and severity):
see "details on results"
CLINICAL SIGNS AND MORTALITY:
No mortality occurred during the study period.
One female at 1000 mg/kg (animal no" 75) showed hunched posture and piloerection from Day 20 post-coitum until Day 3 of lactation. In another female at 1000 mg/kg (animal no. 79) piloerection was noted on Day 4 of lactation. Slight salivation was noted incidentally in males and females treated at 1000 mg/kg. This observation was considered related to the taste of the formulation, and was not considered toxicologically relevant.
All other clinical signs (scabs and alopecia) were considered unrelated to treatment

BODY WEIGHT AND WEIGHT GAIN:
A lower mean body weight and body weight gain was recorded for males at 1000 mg/kg during the complete treatment period. For females a lower mean body weight was noted during the complete treatment period (not always statistically significant) and body weight gain was
decreased on Day 8 of pre mating and Day 1 of mating. Females at 100 and 300 mg/kg showed a slightly lower mean absolute body weight on Day 8 of pre mating. As these had recovered on Day 1 of mating, it was not considered toxicologically significant

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption before or after allowance for body weight was reduced in males and females at 1000 mg/kg during the whole observation period (not always statistically significant). Food consumption at 100 and 300 mg/kg was similar to controls.

HAEMATOLOGY:
A slightly higher white blood cell count was noted in males and females at 1000 mg/kg (not statistically significant for males). In addition, relative numbers of reticulocytes were increased in males at 1000 mg/kg. Other findings reaching statistical significance (increased basophils in males at 100 and 300 mg/kg, increased mean corpuscular volume (MCV) in females at 300 and 1000 mg/kg, increased mean corpuscular haemoglobin (MCH) in females at 300 mg/kg, decreased mean corpuscular haemoglobin concentration (MCHC) in females at 1000 mg/kg) were considered to
be of no toxicological relevance as these findings either remained within the range considered normal for rats of this age and strain or showed no dose response relationship.

CLINICAL BIOCHEMISTRY:
Increased activity of alanine aminotransferase was noted in males at 1000 mg/kg and for two females at 1000 mg/kg. One male at 1000 mg/kg showed increased activity of aspartate aminotransferase. Other findings reaching statistical significance (decreased levels of total bilirubin in males at 100
and 300 mg/kg, increased level of total bilirubin in males at 1000 mg/kg, slightly decreased sodium in males at 1000 mg/kg, and slightly decreased albumin in females at 300 mg/kg) were considered to be of no toxicological relevance in absence of a dose response relationship.

ORGAN WEIGHTS:
Males at 1000 mg/kg showed a lower mean terminal body weight as compared to that of controls. Organ/body weight ratios were increased for liver, spleen, testes and epididymides in males and for liver, spleen, kidneys and adrenals in females at 1000 mg/kg. The increased organ/body weight ratio noted for brain in males at 1000 mg/kg was considered to be caused by the relatively low terminal body weight rather than being a direct effect of
treatment. Absolute brain weight was slightly decreased in females at 1000 mg/kg. However, as this was very slight and the brain weight ratio was unaffected, this finding was not considered toxicologically relevant. No organ weight changes were noted at 100 and 300 mg/kg body weightlday.

GROSS PATHOLOGY:
There were no treatment-related findings. There were no significant changes in the reproductive organs of those animals that had failed to
mate; the ovaries of one rat in Group 3 had apparently not ovulated regularly based on the observation that the corpora lutea were hypercellular suggesting ageing. There was a usual distribution of stage types in the testes stained with PAS. There were occasional observations of commonplace changes in the tissues of young laboratory rats. These were all of small degree and variable incidence. None were associated with treatment.

HISTOPATHOLOGY:
macroscopic findings: no treatment-related findings. Those few observations that were present were those that are commonplace in the tissues of young laboratory rats
microscopic findings: no treatment-related findings.

REPRODUCTION DATA:
Reproduction parameters were unaffected by treatment up to 1000 mg/kg body weightlday. No treatment related findings were observed for mating performance, fertility parameters, duration of gestation and number of dead and living pups at first litter check.

BREEDING DATA:
Breeding parameters were unaffected by treatment up to 1000 mg/kg body weight/day. Postnatal loss and viability index were similar for the control and treated groups.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
haematology
clinical biochemistry
organ weights and organ / body weight ratios
gross pathology
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
dissolved
Sex:
male/female
Basis for effect level:
reproductive function (oestrous cycle)
reproductive function (sperm measures)
reproductive performance
Remarks on result:
other: Generation: definitive reproduction, breeding, development (migrated information)
Clinical signs:
no effects observed
Description (incidence and severity):
see "details on results"
Mortality / viability:
no mortality observed
Description (incidence and severity):
see "details on results"
Body weight and weight changes:
no effects observed
Description (incidence and severity):
see "details on results"
Sexual maturation:
no effects observed
Description (incidence and severity):
see "details on results"
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
see "details on results"
Gross pathological findings:
no effects observed
Description (incidence and severity):
see "details on results"
Histopathological findings:
no effects observed
Description (incidence and severity):
see "details on results"
VIABILITY (OFFSPRING):
Development of pups was unaffected by treatment up to 1,000 mg/kg bw/day.

CLINICAL SIGNS (OFFSPRING):
Incidental clinical symptoms consisted of scabs, discolouration tail apex, and blue spot neck. Incidental macroscopic findings consisted of wound at the head, scabs at the neck, and dark red discolouration of the tail apex. No relationship with treatment was established for these observations and they were considered to be within the normal biological variation for rats of this age and strain.

BODY WEIGHT (OFFSPRING):
(Mean) body weights were similar for the control and treated groups.

GROSS PATHOLOGY (OFFSPRING):
All offspring was sexed and externally examined. The stomach was examined for the presence of milk. Descriptions of all external abnormalities were recorded. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue was preserved in 10% buffered formalin, for possible further examination.
Remarks on result:
not measured/tested
Reproductive effects observed:
not specified
Conclusions:
In conclusion, treatment with the test substance by oral gavage in male and female rats at dose levels of 100, 300 and 1,000 mg/kg bw./d revealed parental toxicity at 1,000 mg/kg bw/d. No reproduction, breeding and developmental toxicity was observed for treatment up to 1,000 mg/kg bw/d. Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established at 300 mg/kg. The definitive reproduction, breeding and developmental NOAEL was established as being 1,000 mg/kg.
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

Short description of key information:
OECD 422:

In conclusion, treatment with the test substance by oral gavage in male and female rats at dose levels of 100, 300 and 1,000 mg/kg bw./d revealed parental toxicity at 1,000 mg/kg bw/d. No reproduction, breeding and developmental toxicity was observed for treatment up to 1,000 mg/kg bw/d. Based on these findings, the parental No Observed Adverse Effect Level (NOAEL) was established at 300 mg/kg. The definitive reproduction, breeding and developmental NOAEL was established as being 1,000 mg/kg.

Justification for selection of Effect on fertility via oral route:
OECD 422

Effects on developmental toxicity

Description of key information
On the basis of the above results, the dosage of 300 mg/kg/day is considered the NOAEL for females. No NOAEL can be defined for foetuses.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2013 to 27 July 2013
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rat
Strain:
Wistar
Details on test animals and environmental conditions:
A total of 120 Wistar Hannover (Hsd Brl Han: WIST) virgin female rats, 10 weeks old (weight range of 171-209 g) were received from Harlan
Italy s.r.l., San Pietro al Natisone (UD), Italy. The 48 male rats used were from the same supplier and were at least 11 weeks old (weight
range of 339-375 g). After arrival the weight range was determined and the animals were uniquely identified by tattoo on the hind feet. A
health check was then performed by a veterinarian. An acclimatisation period of 19 days was allowed before the start of treatment, during
which time the health status of the animals was assessed by thorough observations.
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22
°C/pm 2 °C and 55%\pm 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from
these ranges were recorded. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours
each day.
Route of administration:
oral: gavage
Vehicle:
other: purified water
Details on exposure:
The required amount of Sodium Hydroxymethansulfinate was dissolved in the vehicle (purified water). The formulations were prepared daily
(concentrations of 10, 30 and 100 mg/mL) and the concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the
same dose volume.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable (content
check). Results of the analyses were within the limits of acceptance and the stability was found to be 24 hours at room temperature and 8
days at +4°C and verified in RTC Study No.: 95740. Samples of the formulations prepared on Week 1 and last Week (when all females from
different groups were present) were also analysed to check the concentration. Results of the analyses were within the limits of acceptance.
Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. 95740), in
the range from 5 to 200 mg/mL.
The software used for this activity was Empower® Pro build No. 2154.
Details on mating procedure:
The females were paired with male rats. Females were paired one to one in the home cage of the male and left overnight. Vaginal smears
were taken daily in the morning from the day after pairing until a positive identification of mating was made. The day of mating, as judged by
the presence of sperm in the vaginal smear or by the presence of a copulation plug, was considered as Day 0 of gestation (or Day 0 post
coitum).
Duration of treatment / exposure:
All animals were dosed once a day, from Day 6 through Day 19 post coitum.
Frequency of treatment:
daily
Duration of test:
20 days
No. of animals per sex per dose:
24 mated females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Dose levels were selected in consultation with the Sponsor based on information from previous studies.
Maternal examinations:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a
similar procedure was followed except that the final check was carried out at approximately mid-day. Pups were found on the cage tray of
female no. 95760137 on Day 13 post coitum. The female and pups were sacrificed the day of occurrence. A complete necropsy was
performed.

Clinical signs
All clinical signs were recorded for individual animals. Each animal was observed at least once daily and any clinical signs recorded starting
from allocation until sacrifice.

Body weight
All animals were weighed on Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

Food consumption
Food consumption was measured on Days 3, 6, 9, 12, 15, 18 and 20 post coitum, starting from Day 0 post coitum.

Euthanasia
The animals were killed on Day 20 post coitum and necropsied. All females were euthanised with carbon dioxide. All foetuses were sacrificed
by intraperitoneal injection of Sodium Thiopental followed by hypothermia. All animals were subjected to necropsy.

Necropsy
The clinical history of the females was studied and a detailed post mortem examination was conducted (including examination of the external
surface and orifices). Changes were noted and the abnormalities preserved in 10% neutral buffered formalin.
Ovaries and uterine content:
The ovaries and uteri were examined to determine:
• gravid uterine weight (not obtained from female no. 95760137 sacrificed during the study);
• number of corpora lutea;
• number of implantation sites;
• number, sex and weight of all live foetuses;
• number and sex of dead foetuses (foetuses at term without spontaneous movements and breathing);
• number of intra-uterine deaths;
• gross evaluation of placentae.

Intra-uterine deaths were classified as:
• Early resorptions: only placental remnants visible.
• Late resorptions: placental and foetal remnants visible.

Uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of
embryonic death at very early stages of implantation.
Fetal examinations:
All live foetuses were examined externally. Approximately one-half of the foetuses (i.e., routinely, every second live foetus) in each litter were
preserved in Bouin's solution for subsequent fixed-visceral examination. The remaining foetuses were eviscerated after which the carcasses
were fixed in 95% (v/v) ethanol for subsequent skeletal examination after bone staining with Alizarin Red S. Skeletal and fixed-visceral
examinations were performed in all groups. Structural deviations were classified as follows:

Malformations
Major abnormalities that are rare and/or affect the survival or health of the species under investigation.

Anomalies
Minor abnormalities that are detected relatively frequently.

Variants
A change that occurs within the normal population under investigation and is unlikely to adversely affect survival or health. This might include
a delay in growth or morphogenesis that would have otherwise followed a normal pattern of development.
Statistics:
For body weight, body weight gain and food consumption, the significance of the differences amongst group means was assessed by
Dunnett's test or a modified t test, depending on the homogeneity of data. Statistical analysis of litter data and sex ratio, terminal body weight,
gravid uterus weight and absolute weight gain was carried out by means of the Kruskal-Wallis test and intergroup differences between the
control and treated groups assessed by a non-parametric version of the Williams test.
Indices:
Pre-implantation loss was calculated as a percentage from the formula:
Pre impl. Loss % = no. of corpora lutea-no. of implantations \ no. of corpora lutea X 100
Post-implantation loss was calculated as a percentage from the formula:
Post impl. Loss % = no. of implantations-no. of live foetuses \ no. of implantations X 100
Total implantation loss was calculated as a percentage from the formula:
Total impl. Loss % = no. of corpora lutea-no. of live foetuses \ no. of corpora lutea X 100
Sex ratios of the foetuses were calculated as the percentage of males
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Body weight and body weight gain
A slight (4-5%) statistically significant decrease in body weight was observed from Day 15 to Day 20 of gestation in high dose females (1000
mg/kg/day). No toxicological relevant changes were seen in body weight in low and mid-dose females (100 and 300 mg/kg/day).
No toxicological significance was attributed to the slight increase (statistically significant) in body weight gain observed in females receiving
100 mg/kg/day on Day 12 of gestation and in females receiving 300 mg/kg/day on Day 6 of gestation (before treatment start).
Moreover, a statistically significant decrease in body weight gain (-25 to -81%) was observed in high dose females receiving 1000 mg/kg/day
on Days 9, 15 and 20 of gestation.

Food consumption
A decrease in food consumption (-19 to -22%), with statistical significance on Days 9 and 12 of post coitum was recorded in high dose females
(1000 mg/kg/day) starting from Day 9 of post coitum. No relevant effects on food consumption were seen in low and mid-dose females (100
and 300 mg/kg/day).
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Skeletal examination of foetuses
At skeletal examination the following malformations in foetuses from different litters were observed:
- pubis unossified was seen at 100 mg/kg/day: in 2 foetuses (1.53% of foetuses and 4.55% of litters); at 300 mg/kg/day: in 5 foetuses (4.24%
of foetuses and 10.00% of litters) and at 1000 mg/kg/day: in 2 foetuses (1.41% of foetuses and 8.70% of litters);
- ischium unossified was seen in at 300 mg/kg/day: in 1 foetus (0.85% of foetuses and 5.00% of litters) and at 1000 mg/kg/day: in 1 foetus
(0.70% of foetuses and 4.35% of litters).
These malformations are not present in our historical control data.
An increased presence of additional rudimentary 14th rib (variant) was noted in all groups, incomplete ossification of sternebrae (variant) was
noted in the mid- and high dose groups (300 and 1000 mg/kg/day), metatarsals of the hind paw incompletely or unossified (anomaly) were
observed in all treated groups.

Visceral examination of foetuses
At visceral examination the following malformations in foetuses from different litters were observed:
- anophtalmia was seen at 300 mg/kg/day: in 2 foetuses (1.80% of foetuses and 10.00% of litters) and at 1000 mg/kg/day: in 1 foetus (0.78%
of foetuses and 4.35% of litters);
- hydrourether was seen at 1000 mg/kg/day: in 2 foetuses (1.56% of foetuses and 8.70% of litters);
- umbilical hernia was seen at 1000 mg/kg/day: in 1 foetus (0.78% of foetuses and 4.35% of litters).
No malformations were noted in foetuses of the low dose group (100 mg/kg/day).
Dose descriptor:
NOAEL
Remarks:
(for foetuses)
Basis for effect level:
other: teratogenicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
On the basis of the above results, the dosage of 300 mg/kg/day is considered the NOAEL for females. No NOAEL can be defined for foetuses.
Executive summary:

Study design

The purpose of this study was to investigate the effects of Sodium Hydroxymethansulfinate on pregnancy and embryo-foetal development in the Wistar rat, following oral administration, at dose levels of 100, 300 and 1000 mg/kg/day.

Females were dosed starting from Day 6 until Day 19 post coitum. The test item was administered, by the oral route, at a dose volume of 10 mL/kg. The vehicle was purified water. The following investigations were performed: mortality check, clinical signs, body weight, food consumption and macroscopic observation of the females. On Day 20 post coitum at necropsy, gravid uterus weight, corpora lutea, implantation sites, number, sex and weight of all foetuses were determined. Gross evaluation of the placenta and external examination on all foetuses were also performed. Fixed-visceral and skeletal examinations were performed on foetuses of all groups.

Fate of females

No deaths related to treatment occurred. The number of females with live foetuses on gestation Day 20 was 21 in the control group, 22 in the low dose group, 20 in the mid-dose group and 23 in the high dose group. One female in the mid-dose group was sacrificed on Day 13 post coitum with litter since pups were found on the cage tray.

Clinical signs

No signs related to treatment, nor signs of reaction to treatment were observed during the dosing period.

Body weight and body weight gain

A slight statistically significant decrease in body weight was observed from Day 15 to Day 20 of gestation in high dose females (receiving 1000 mg/kg/day).

No toxicological relevant changes were seen in body weight in low and mid-dose females (100 and 300 mg/kg/day).

No toxicological significance was attributed to the slight increase (statisticallysignificant) in body weight gain observed in females receiving 100 mg/kg/day on Day 12 of gestation and in females receiving 300 mg/kg/day on Day 6 of gestation (before treatment start).

In addition, a statistically significant decrease in body weight gain was observed on Days 9, 15 and 20 of gestation in the high dose group (1000 mg/kg/day).

Food consumption

A decrease in food consumption with statistical significance on Days 9 and 12 of post coitum was recorded in high dose females (receiving 1000 mg/kg/day), starting from Day 9 of post coitum to the end of treatment.

No relevant effects on food consumption were seen in low and mid-dose females (100 and 300 mg/kg/day).

Terminal body weight, uterus weight and absolute weight gain

Terminal body weight of high dose females was reduced compared to controls and a statistically significant decrease in absolute weight gain (terminal body weight at necropsy minus gravid uterine weight, minus body weight at Day 0 post coitum) was also noted.

Litter data and sex ratios

Litter data, mean foetal weight and sex ratios were not affected by treatment.

Macroscopic examination

No treatment-related changes were observed at post mortem examination in treated animals, when compared to the controls.

External examination of foetuses

Small foetuses were present in all groups including controls with similar incidence.

Skeletal examination of foetuses

At skeletal examination the following malformations in foetuses from different litters were observed:

- pubis unossified was seen in 2 foetuses in the low dose group (100 mg/kg/day) corresponding to 1.53% of foetuses and 4.55% of litters, in 5 foetuses in the mid-dose group (300 mg/kg/day) corresponding to 4.24% of foetuses and 10.00% of litters and in 2 foetuses in the high dose group (1000 mg/kg/day) corresponding to 1.41% of foetuses and 8.70% of litters;

-ischium unossified was seen in 1 foetus in the mid-dose group (300 mg/kg/day) corresponding to 0.85% of foetuses and 5.00% of litters and in 1 foetus in the high dose group (1000 mg/kg/day) corresponding to 0.70% of foetuses and 4.35% of litters.

Pubis and ischium unossified are not present in the historical control data.

An increased presence of additional rudimentary 14th rib (variant) was noted in all groups with particular emphasis in the low dose and high dose groups (100 and 1000 mg/kg/day), incomplete ossification of sternebrae (variant) was noted in the mid- and high dose groups (300 and 1000 mg/kg/day), metatarsals of the hind paw incompletely or unossified (anomaly) were observed in all treated group with particular emphasis in the mid-dose group (300 mg/kg/day).

Visceral examination of foetuses

At visceral examination the following malformations in foetuses from different litters were observed:

- anophtalmia was seen in 2 foetuses in the mid-dose group (300 mg/kg/day) corresponding to 1.80% of foetuses and 10.00% of litters and in

1 foetus in the high dose (1000 mg/kg/day) corresponding to 0.78% of foetuses and 4.35% of litters.

- hydrourether was seen in 2 foetuses in the high dose group (1000 mg/kg/day) corresponding to 1.56% of foetuses and 8.70% of litters.

-umbilical hernia was seen in 1 foetus in the high dose (1000 mg/kg/day) corresponding to 0.78% of foetuses and 4.35% of litters.

No malformations were noted in foetuses of the low dose group (100 mg/kg/day).

As a consequence of the findings, a classification of the substance into the hazard class “Reproductive toxicity”, Category 2 (H361d)

according to the Regulation (EC) No. 1272/2008 is recommended.

Based on the experimental results, the criteria for classification into Category 1 are not met. However, the above mentioned adverse effects on pup development should be considered. Taking into account of the actual substance’s classification into the hazard class “Germ cell mutagenicity” category 2, which is based on test data of the substance, a classification of the substance as “reproductive toxic, Category 2; H361d” appears to be appropriate.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Additional information

As a consequence of the findings, a classification of the substance into the hazard class “Reproductive toxicity”, Category 2 (H361d)

according to the Regulation (EC) No. 1272/2008 is recommended.

Based on the experimental results, the criteria for classification into Category 1 are not met. However, the above mentioned adverse effects on pup development should be considered. Taking into account of the actual substance’s classification into the hazard class “Germ cell mutagenicity” category 2, which is based on test data of the substance, a classification of the substance as “reproductive toxic, Category 2; H361d” appears to be appropriate.


Justification for selection of Effect on developmental toxicity: via oral route:
OECD 414

Justification for classification or non-classification

As a consequence of the findings, a classification of the substance into the hazard class “Reproductive toxicity”, Category 2 (H361d)

according to the Regulation (EC) No. 1272/2008 is recommended.

Based on the experimental results, the criteria for classification into Category 1 are not met. However, the above mentioned adverse effects on pup development should be considered. Taking into account of the actual substance’s classification into the hazard class “Germ cell mutagenicity” category 2, which is based on test data of the substance, a classification of the substance as “reproductive toxic, Category 2; H361d” appears to be appropriate.