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Key value for chemical safety assessment

Effects on fertility

Description of key information

A key combined toxicity and reproductive screening toxicity study according to OECD 422 guideline was available for read across substance butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268 -36 -6). The test item was administered orally to rats at dose levels of 60, 120 or 300 mg test item/kg bw/day. Systemic toxicity effects were seen at 120 and 300 mg/kg bw/day. At 300 mg/kg bw/day, a high loss of implantation sites in the high dose group led to a statistically significant reduction in the gestation index, in the mean number of implantation sites per dam, in the mean number of born pups per dam and in the birth index. Accordingly, the implantation loss index was statistically significantly increased in the high dose group. The high percentage of stillbirths led to a statistically significantly reduced live birth index in the high dose group. Test item related effects were also noted on the pups from the 3 remaining dams of the high dose group, expressed by a statistically significantly reduced survival rate during the lactation period, a statistically significant reduction in the mean litter weight and in the total litter weight per dam on lactation day 1 and 4. In conclusion, the NOAEL for reproductive toxicity was 120 mg/kg bw/day. The findings seen at 300 mg/kg bw were considered to be secondary to the paternal and maternal toxicity. The same results were considered to be valid for the Registered substance.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See attached read-across justification
Reason / purpose:
read-across source
Reason / purpose:
reference to same study
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1 male and 1 female of the high dose group died on day 33 and day 26 respectively; slight signs of systemic toxicity were noted predominantly in form of pilo-erection and increased salivation in males and females dosed at 120 & 300 mg/kg bw
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
decreased at 120 and 300 mg/kg bw in male rats and at 300 mg/kg bw in female rats premating, mating, during gestation and during lactation
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
decreased at 120 and 300 mg/kg bw in male rats and at 300 mg/kg bw in female rats premating, mating, during gestation and during lactation
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
hepatocellular hypertrophy and macrovesicular vacuolation in liver and squamous cell hyperplasia in the non-glandular stomach in males and females at 300 mg/kg bw; changes in the mammary glands, the uterus and vagina in females at 300 mg/kg bw
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Test substance intake: actual ingestion
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
reduced gestation index, No. implantation sites and No.(live) born pups per dam and birth index; increased implantation loss index at 300 mg/kg bw
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One of 10 male animals (no. 64) of the high dose group (300 mg test item/kg bw/day) died on test day 33, showing piloerection and reduced motility before death.
One of 10 female animals (no. 71) of the high dose group (300 mg test item/kg b.w./day) died on gestation day 9, showing piloerection and salivation on a few days during the premating, mating and gestation period.
Male animals
Slight salivation was noted in 2 animals of the intermediate dose group (120 mg test item/kg b.w./day) on one day each.
In the high dose group (300 mg test item/kg bw/day) piloerection and slight to moderate salivation was noted for several animals during the whole study for 1 up to 13 test days. Breathing sounds were noted in animal no. 61 for 1 day.
Female animals
In the intermediate dose group (120 mg test item/kg bw/day) piloerection was noted for 1 animal on 1 day during the mating period. During the gestation period moderate salivation was noted in 3 animals on one day each.
In the high dose group (300 mg test item/kg bw/day) piloerection was noted for several animals on 3 up to 10 test days and slight to extreme salivation in all female animals (2 up to 11 test days) during the pre-mating, mating and gestation period. A haemorrhagic vagina or nose was noted for animal no. 72 on gestation days 11 and 16. During the lactation period piloerection, reduced motility and changes in the status of faeces were noted for the emaciated female animal no. 75.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Male animals
A reduction in body weight was noted in the intermediate dose group (120 mg test item/kg bw/day) from test day 8 by 6.7% until the end of the study by 6.2%, statistically significant (p≤0.05) on test days 8, 22, 29 and 42.
In the high dose group (300 mg test item/kg bw/day) the reduction in body weight was more pronounced, with 9.0% on test day 8 and 13.3% at the end of the study, statistically significant (p≤0.01) from test day 8 to the end of the study.
Accordingly, statistically significant (p≤0.05 or p≤0.01) reductions in body weight gain were noted in the intermediate and the high dose group (120 and 300 mg test item/kg bw/day).
Female animals
A decrease in body weight (300 mg test item/kg bw/day) was noted in the high dose group, starting at the end of the pre-mating period by 5.4%. Statistically significant (p≤0.01) reductions were noted during the gestation period from gestation day 7 (by 9.6%) to 20 (by 23.9%) and on lactation day 1 (by 22.0%) and 4 (by 23.8%).
Statistically significant reductions in body weight gain were noted on gestation day 14 (p≤0.01) and 20 (p≤0.05) in the intermediate dose group (120 mg test item/kg bw/day).
In the high dose group (300 mg test item/kg bw/day) statistically significant reductions in body weight gain were noted on test day 8 (p≤0.01) and during the gestation period from gestation day 7 (p≤0.05) to gestation day 20 (p≤0.01).
Male animals
A statistically significant (p≤0.01) reduction in food consumption by 14.1% was noted in the intermediate dose group (120 mg test item/kg bw/day) during the first test week.
In the high dose group (300 mg test item/kg bw/day) the food consumption was statistically significantly (p≤0.01) reduced by 20.7% during the first and by 15.3% during the second test week.
Female animals
At the intermediate dose group (120 mg test item/kg bw/day) a statistically significant reduction in food consumption was noted during the first week of gestation by 7.3% (p≤0.05) and during the second week of gestation by 13.8% (p≤0.01).
In the high dose group (300 mg test item/kg bw/day) a statistically significant (p≤0.01) reduction in food consumption was noted from the first test week (by 26.3%) until the end of the gestation period (by 6.8%).

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): gavage

REPRODUCTIVE FUNCTION:
-Pre-coital time:
No test item-related influence was noted.
-Gestation length:
No test item-related influence was noted.
-Evaluation of reproduction parameters of the dams:
Only 3 of 8 pregnant dams of the high dose (300 mg test item/kg) group littered live pups, leading to a statistically significant (p≤0.01) reduced gestation index of 37.5%.
Five of 8 pregnant dams showed a total loss of implantation sites, leading to a statistically significant (p≤0.05) reduction in the mean number of implantation sites per dam and a statistically significantly (p≤0.01) increased post-implantation loss of 66.3%.
Accordingly, the whole number of born pups (alive and dead) per dam and the number of live born pups per dam were statistically significantly (p≤0.01) reduced, leading to a statistically significant (p≤0.01) reduction of the birth index to 39.5%.
The percentage of stillbirths in the high dose group was 14.7%, leading to a statistically significant (p≤0.05) reduction in the live birth index.
No test item related differences were noted for the fertility index and the preimplantation loss.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Male animals
Starting at the intermediate dose group (120 mg test item/kg bw/day) a statistically significant (p≤0.05, right only) dose related decrease by 18.3% at maximum was noted for the absolute organ weight of the left + right epididymis.
In the high dose group (300 mg test item/kg) the following statistically significant changes were noted:
An increase by 34.0% (p≤0.01) of the relative liver weight and by 19.6% (non-significant) of the absolute liver weight.
The relative and absolute organ weight of the thymus was decreased by 42.5% (p≤0.05) and by 49.0% (p≤0.01).
The relative organ weights of the left and right gonads were reduced by 14.9% (p≤0.01) and by 13.7% (p≤0.05).
Female animals
In the intermediate dose group (120 mg test item/kg) a statistically significant (p≤0.01) increase was noted in the relative liver weight by 23%.
The absolute heart weight was statistically significantly decreased by 16.7%.
The relative organ weights of the left and right adrenal glands were statistically significantly (p≤0.01) increased by 26.2% and 24.4%.
In the high dose group (300 mg test item/kg) the relative organ weights of the left and right kidneys were statistically significantly increased by 17.2% (p≤0.05) and by 20.9% (p≤0.01).
The absolute organ weight of the heart was statistically significantly (p≤0.05) decreased by 33.1%.
The absolute organ weight of the right gonad was statistically significantly (p≤0.05) decreased by 37.5% and the absolute organ weight of the left gonad non-significantly by 25.5%.
Similar but not statistically significant increases as in the intermediate dose groups were noted for the relative liver weight and the left and right adrenal glands.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Body weight at autopsy:
Male animals
In the high dose group (300 mg test item/kg bw/day) the body weight at autopsy was statistically significantly (p≤0.01) decreased by 13.5%.
Female animals
A statistically significant (p≤0.01) reduction in the body weight at autopsy by 22.2% was noted in the high dose group (300 mg test item/kg bw/day).
Macroscopic post mortem findings:
Male animals
Macroscopic changes were noted in the stomach of 3 animals of the high dose group (300 mg test item/kg bw/day) in form of a detachment of the mucosa, whitish thickenings and ulcers. The findings were considered to be test item-related.
Female animals
A test item-related detachment of the mucosa was noted in the stomach of one animal of the inter-mediate dose group (120 mg test item/kg bw/day)
In the high dose group (300 mg test item/kg bw/day) a test item-related detachment of the mucosa was noted in 2 animals.

HISTOPATHOLOGY (PARENTAL ANIMALS)(restricted to the control group and the high dose group):
Male and female animals
Test item related changes were noted in the liver (hepatocellular hypertrophy and macrovesicular vacuolation evoked by fatty change) and in the non-glandular stomach (squamous cell hyperplasia) in the animals of the high dose group (300 mg test item/kg).
Female animals
Test item related changes were noted in the mammary glands, the uterus and vagina in form of a decreased acinar development, stromal hyperplasia in the endometrium and metestrus in only 1 of 5 animals of the high dose group (300 mg test item/kg bw/day).

No microscopic changes were noted for the reroductive organs of the male and female rats of the high dose group (300 mg test item/kg bw/day) and no changes were noted on the stages of spermatogenesis.


Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: reproductive effects
Clinical signs:
not specified
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
reduced survival rate of the pups (75.9% versus control) in the high dose group of 300 mg test item/kg
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced at 300 mg/kg bw on lactation day 1 by 17.1% (p≤0.05) and on lactation day 4 by 29.3% (p≤0.01)
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
The survival rate of the pups was statistically significantly reduced to 75.9% in the high dose group (300 mg test item/kg)

BODY WEIGHT (OFFSPRING)
The mean litter weight of the pups was statistically significantly reduced on lactation day 1 by 17.1% (p≤0.05) and on lactation day 4 by 29.3% (p≤0.01) in the high dose group (300 mg test item/kg).
The total litter weight per dam was also statistically significantly reduced on lactation day 1 by 42.4% (p≤0.05) and on lactation day 4 by 56.3% (p≤0.01) in the high dose group (300 mg test item/kg).

GROSS PATHOLOGY (OFFSPRING)
No visible gross abnormalities were noted in the control and the treatment groups.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
120 mg/kg bw/day
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
viability
other: decreased pup number and body weights
Reproductive effects observed:
not specified

Table 1. Fertility and Reproductive parameters Parental generation

Parameter

Group 1

Control

Group 2

60 mg/kg

Group 3

120 mg/kg

Group 4

300 mg/kg

No. females evaluated

10

10

10

10

Mean precoital time (days)

3.8

3.4

2.9

3.8

Number of pregnant dams

10

10

10

8 +1#

Fertility index (%)

100

100

100

90

No. dams with pups (live + dead)

10

10

10

4

Gestation length (days)

22.7

22.9

22.9

23.3

No. dams with live pups

10

10

10

3

Gestation Index (%)

100

100

100

37.5**

No.Corpora lutea(total)

160

142

151

104

No.Corpora lutea(mean)

16.0

14.2

15.1

13.0

No. Implantation sites (total)

143

119

149

86

No. Implantation sites (mean)

14.3

11.9

14.9

10.8v

Number of pups at birth (total)

136

109

134

34vv

Number of pups at birth (mean)

13.6

10.9

13.4

4.3vv

Birth Index (mean %)

95.1

90.3

90.2

36.0

Birth Index (total %)

95.1

91.6

89.9

39.52

Number of stillbirths

3

0

4

5

No. of dams with stillborn pups

2

0

1

2

Number of live born pups (total)

133

109

130

29vv

Number of live born pups (mean)

13.3

10.9

13.0

3.6vv

Live birth index (mean %)

97.7

100.0

96.7

67.3

Live birth index (total %)

97.8

100.0

97.0

85.3b

Pre-implantation loss (mean %)

8.8

16.1

1.3

14.7

Pre-implantation loss (total %)

10.6

16.2

1.3b

17.3

Post-implantation loss (mean %)

7.9

9.7

12.6

68.5

Post-implantation loss (total %)

7.0

8.4

12.8

66.3b

1 p≤0.05 Chi2-test

2 p≤0.01 Chi2-test

*  p≤0.05 Fisher test

**p≤0.01 Fisher test

v p≤0.05 Dunnett test or Student’s t-test

vvp≤0.01 Dunnett test or Student’s t-test

ap< 0.05 Chi2-test

bp<0.01 Chi2-test

#: Animal no. 71 was excluded, because of its premature death on gestation day 9

Conclusions:
The following no-observed adverse-effect levels were established:
Paternal and Maternal toxicity: NOAEL= 60 mg/kg b.w./day, p.o.
Reproductive toxicity: NOAEL= 120 mg/kg b.w./day, p.o.
Executive summary:

The aim of the study was to obtain information on possible effects of the read-across subsance butanedioic acid, sulfo-, 1-C12-18-alkyl esters, disodium salts (CAS 90268 -36 -3) when administered orally to rats at dose levels of 60, 120 or 300 mg test item/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 43 for the male rats and on lactation day 4 or shortly thereafter for the female rats.was administered orally to rats at dose levels of 60, 120 or 300 mg test item/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 43 for the male rats and on lactation day 4 or shortly thereafter for the female rats.

Effects on the parental generation (general toxicity) :

One of 10 male and one of 10 female animals of the high dose group (300 mg test item/kg bw/day) died prematurely on test day 33 or on gestation day 9 (TD 26). Slight to moderate salivation was noted in a few male and female animals of the intermediate dose group (120 mg test item/kg bw/day) on 1 day each, which was regarded as test item-related. In the high dose group (300 mg test item/kg bw/day) piloerection and a slight to extreme salivation was noted for several to all male and female animals on several days and regarded as test item-related. A statistically significant reduction in body weight was noted for the male animals of the intermediate dose group (120 mg test item/kg bw/day) and for both sexes at the high dose group (300 mg test item/kg bw/day).

Statistically significant increases in the activity of ALAT and/or aP and ASAT and decreases in the globulin, cholesterol, chloride and potassium concentrations were noted for the male and/or female animals of the intermediate and/or the high dose group (120 and/or 300 mg test item/kg bw/day).

Statistically significant changes were noted for several organ weights of the male and female animals of the intermediate and the high dose group (120 and 300 mg test item/kg bw/day), most remarkable for the thymus and liver weights of the animals of the high dose group.

Macroscopic inspection at autopsy revealed test item-related changes in the stomachs of male and female animals of the intermediate and high dose group (120 and 300 mg test item/kg bw/day).

Histopathological examination of the organs from animals of the high dose group (300 mg test item/kg bw/day) revealed test item-related changes in the liver (hepatocellular hypertrophy and macrovesicular vacuolation evoked by fatty change) and the non-glandular stomach (squamous cell hyperplasia) of male and female animals.

Reproductive toxicity

The high number of 5 pregnant dams with a total loss of implantation sites in the high dose group (300 mg test item/kg bw/day) led to a statistically significant reduction in the gestation index, in the mean number of implantation sites per dam, in the mean number of born pups per dam and in the birth index. Accordingly, the implantation loss index was statistically significantly increased in the high dose group.

The high percentage of stillbirths led to a statistically significantly reduced live birth index in the high dose group (300 mg test item/kg bw/day). Test item related effects were also noted on the pups from the 3 remaining dams of the high dose group (300 mg test item/kg bw/day), expressed by a statistically significantly reduced survival rate during the lactation period, a statistically significant reduction in the mean litter weight and in the total litter weight per dam on lactation day 1 and 4.

The following no-observed adverse-effect levels were established:

Paternal and Maternal toxicity: NOAEL= 60 mg/kg b.w./day, p.o.; Reproductive toxicity: NOAEL=120 mg/kg b.w./day, p.o.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
120 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
reliable
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A key combined toxicity and reproductive screening toxicity study according to OECD 422 guideline was available for read across substance CAS 90268 -36 -6 (Hansen, 2013). The test item was administered orally to rats at dose levels of 60, 120 or 300 mg test item/kg bw/day. The application started two weeks before mating on test day one and ended on the day or one day before sacrifice. Day of sacrifice was on test day 43 for the male rats and on lactation day 4 or shortly thereafter for the female rats. Systemic toxicity effects are described under Section 7.5.1, where it was evident that 120 and 300 mg/kg bw were showing paternal and maternal toxicity.

The high number of 5 pregnant dams with a total loss of implantation sites in the high dose group (300 mg test item/kg bw/day) led to a statistically significant reduction in the gestation index, in the mean number of implantation sites per dam, in the mean number of born pups per dam and in the birth index. Accordingly, the implantation loss index was statistically significantly increased in the high dose group.

The high percentage of stillbirths led to a statistically significantly reduced live birth index in the high dose group (300 mg test item/kg bw/day). Test item related effects were also noted on the pups from the 3 remaining dams of the high dose group (300 mg test item/kg bw/day), expressed by a statistically significantly reduced survival rate during the lactation period, a statistically significant reduction in the mean litter weight and in the total litter weight per dam on lactation day 1 and 4. In conclusion, the NOAEL for reproductive toxicity was 120 mg/kg bw/day.

 

A supporting multi-generation study was performed with registered substance similar to OECD 414 and 416. Male and female rats (20/sex) were exposed to AEROSOL® A-268 via feed at 0%, 1% or 4% from weaning until reproductive age. Rats were mated several times: first litter was sacrificed at weaning, the second litter was allowed to mature and the rats were sacrificed at day 13 (10/20) or day 20 (10/20) of the third pregnancy. The first litter of the F1 generation was sacrificed at weaning, F1 rats were sacrificed at day 13 and 20 of the second pregnancy (10 rats/ timepoint). No substance-related mortality occurred in the parental animals of P and F1 generation, no substance-related histopathology was found. The fertility index was decreased for the highest concentration group calculated for both litters of the P generation, but not for the first litter of the second generation. The number of viable fetuses (average/ litter) was decreased in a dose-related manner for all litters. This corresponds to a dose-related decrease in the number of implantation sites in both exposed groups seen in rats sacrificed at day 13 of pregnancy. The viability of the pups until weaning was decreased in the first litters of the P and F1 generation at the highest dose only. The average weaning weight of pups was decreased (at least with 10%) at both dose levels in all litters. Based on these results, a LOAEL for reproduction and development of 1% A-268 was established (corresponding to a dose level of appr. 500 mg/kg bw/ day) in absence of systemic toxicity of the parents. Based on the available data, the LOAEL of disodium 4-isodecyl sulfosuccinate is estimated to be 475 mg/ kg bw/ day.

Justification for classification or non-classification

The available data do not indicate that the Registered subsance is reprotoxic The substance is thus not classified for reprotoxic properties according to CLP Regulation (EC) No. 1272/2008.