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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Identity Sodium Hydroxymethansulfinate
Trade name Bruggolite[superscript:®] E02
Chemical names Sodium hydroxymethanesulfinate dihydrate, Hydroxymethane sulfinic acid, monosodium salt
Label name Bruggolite E02 Pulver
Batch no. 13042401
Expiry date 30 April 2014
Storage conditions room temperature, protected from humidity
CAS Number 149-44-0
EC Number 205-739-4
RTC number 13647

Test animals

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.

Administration / exposure

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.

Examinations

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

Results and discussion

Results: maternal animals

Maternal developmental toxicity

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).

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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).

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks:
(for foetuses)
Basis for effect level:
other: teratogenicity
Remarks on result:
not determinable
Remarks:
no NOAEL identified

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

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.