Hexasodium 2,2'-[vinylenebis[(3-sulphonato-4,1-phenylene)imino[6-[bis(2-hydroxyethyl)amino]-1,3,5-triazine-4,2-diyl]imino]]bis(benzene-1,4-disulphonate)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

NOAEL for fertility = 250 mg/kg bw/day (based on the OECD 422 study on OB 3a-DSA)

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

250 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance under registration OB 3a-DSA belongs to the category of Stilbene Fluorescent Whitening Agents. The reproductive toxicological potential was firstly assessed through Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening (OECD 422) carried out on the substance under registration. Details on the Read Across approach for category are reported in a document attached in IUCLID section 13.

Repeated oral administration at doses of 80, 250 and 750 mg/kg bw/day to rats by gavage did not cause the death of animals. Test item treatment did not produce clinical changes in health status of animals, did not affect the normal growth of males and females.

During the biometry of reproductive organs statistically significantly increased absolute and relative weight of prostate gland with seminal vesicles in males at the middle and highest dose levels was found out. During biometry of reproductive organs in females statistically significantly decreased absolute weight of ovaries was recorded.  Relative weight of thyroid gland was statistically significantly increased in females in the highest dose level.  

No findings related with the test item treatment were detected during the examination of microscopical structure of reproductive organs in animals at the highest dose level. In females, histopathological findings in uterus related to females’ gravidity were found out.  

Examination of sperm motility and sperm morphology did not show any significant changes in males.

Concentration of hormones T4 and TSH in males from treated groups was similar with the control group.  During the microscopical evaluation of thyroid gland in males there were no findings.

Concentration of hormones T4 and TSH in pups from treated groups was similar with the control group.  The weight of the thyroid gland was not significantly changed in male and female pups of treated mothers in comparison with the control group of mothers.

During the microscopical evaluation of thyroid gland in pups no changes were found out.

Number of females achieving pregnancy was decreased in the middle and highest dose levels. Stillborn pups were detected in one mother from the highest dose level (all litter). Effect of cannibalism of pups in female was observed in the lowest and highest doses as well as in the control group.

Male and female fertility index and was decreased in the middle and highest dose levels. The gestation index was decreased in females at the lowest and highest dose levels. The post-natal losses were not significantly changed in treated and control groups. Markedly increased post-implantation losses in females in the highest dose level were recorded.

The total number of pups was decreased in females in all treated groups, but in the highest dose level decreased total number of pups was marked and with statistical and biological significance.

The decreasing of total number of pups and increasing of post-implantation losses were marked in the highest dose level and can be considered as biologically significant.

The weight of litters was statistically significantly decreased in highest dose level at all examination intervals and in the lowest and middle dose levels at the end of lactation period. The weight of pups was not changed compared to control group. No presence of nipples in male pups was recorded and anogenital distance in treated male and female pups was not biologically significantly affected. No macroscopical findings in pups were found out during pathological examination.

Accordingly, the NOAEL for reproduction was established as 250 mg/kg body weight/day. The NOAEL was established on the based marked increased post-implantation losses and marked decreased total number of pups in females in the highest dose level. While, the NOAEL for development was established as 750 mg/kg body weight/day, indeed all changes in developmental parameters of pups observed at all dose levels were considered to be of no toxicological significance.

A further Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening is available on OB 2-DSA and is used within a weight of evidence approach.

The test item was dosed to 150, 300 and 600 mg/kg/bw to rats and no toxicologically adverse effects on growth of animals, food consumption and health condition of animals were detected in both sexes.

The test item administration did not affect male ability to produce sperm that can fertilise eggs and ability of females to become pregnant.  

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in males. Decreased concentration of T4 hormone in males at the dose level 150 mg/kg/day was considered toxicologically insignificant.

The body weight of parental males and females was not significantly affected by the test item application.

Evaluation of the absolute and relative weight of reproductive organs of male and female, as well as the weight of pituitary and thyroid gland revealed only sporadic findings. The absolute weight of testes and epididymis was significantly decreased in males at the dose level 600 mg/kg/day (also reduced absolute weight of testes was noted in males at the dose level 150 mg/kg/day). Evaluation of relative weight of reproductive organs in males, absolute and relative weights of reproductive organs in treated females did not reveal any statistically significant differences compared to control.

Histological examination did not reveal negative effect of the test item on collected reproductive organs, pituitary and thyroid glands.

Spermatogenesis in the testes of the high dose administered males was without any pathological findings. Epididymides of both control and high dose males were without any pathological findings. Sporadic findings were of spontaneous origin.

Examination of sperm motility and morphology in treated parental males did not show any differences in comparison with the control males.

The number of implantations was comparable between treated and control groups.

The calculated parameters (mating and fertility indexes) were similar in all treated groups in comparison with the control groups. Fertility indexes were slightly lower in high dosed groups in comparison with the control group due to one non-pregnant female. Gestation indexes in treated groups were identical with the control group. The viability index was the best in the highest dosed group of females.

The test item did not affect the number of pups and their development.

The total number of pups, mean number of pups per litter and mean litter weight at first litter check after parturition and during the next intervals were similar or higher in treated groups of mothers compared to control group. The mean pup body weights at all checking intervals was slightly lower in all treated groups compared to control group due to higher number of pups per group of treated females. Macroscopical examination of pups did not show negative effect of the test item administration on development of pups.  

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in pups. Male nipple retention at day 13 was not recorded in any male pup. Corrected anogenital distance in treated pups was comparable to the control pups.  

Based on these findings, the NOAEL value for reproduction and development was established as 600 mg/kg/day since no biologically significant changes relevant to reproduction and development were observed.

In conclusion, the similar substances did not show toxicological effects on the develpomental parameters, while an unclear behavior concerning the reproductive aspects was highlighted for the disulfonated subcategory.

In this respect, a conservative value was selected from the study carried out on OB 3a-DSA, according to OECD TG 422, were the NOAEL was established to be 250 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

NOAEL for developmental toxicity = 600 mg/kg bw/day (based on the OECD 422 study on OB 2-DSA)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

The substance under registration OB 3a-DSA belongs to the category of Stilbene Fluorescent Whitening Agents.  As for substance under registration no study on prenatal developmental toxicity is available. The investigation on developmental toxicity potential was preliminary assessed through Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening (OECD 422) carried out on the substance under registration and on a similar substance with the same sulfonation degree. Secondly, even data from studies of developmental toxicity and teratogenicity on other similar substances belonging to the same category were used to fulfill the data gap in a weight of evidence approach. Details on the Read Across approach for category are reported in a document attached in IUCLID section 13.

Repeated oral administration of the substance under registration at doses of 80, 250 and 750 mg/kg bw/day to rats by gavage did not cause the death of animals. Test item treatment did not produce clinical changes in health status of animals, did not affect the normal growth of males and females.

During the biometry of reproductive organs statistically significantly increased absolute and relative weight of prostate gland with seminal vesicles in males at the middle and highest dose levels was found out. During biometry of reproductive organs in females statistically significantly decreased absolute weight of ovaries was recorded.  Relative weight of thyroid gland was statistically significantly increased in females in the highest dose level.  

No findings related with the test item treatment were detected during the examination of microscopical structure of reproductive organs in animals at the highest dose level. In females, histopathological findings in uterus related to females’ gravidity were found out.  

Examination of sperm motility and sperm morphology did not show any significant changes in males.

Concentration of hormones T4 and TSH in males from treated groups was similar with the control group.  During the microscopical evaluation of thyroid gland in males there were no findings.

Concentration of hormones T4 and TSH in pups from treated groups was similar with the control group.  The weight of the thyroid gland was not significantly changed in male and female pups of treated mothers in comparison with the control group of mothers.

During the microscopical evaluation of thyroid gland in pups no changes were found out.

Number of females achieving pregnancy was decreased in the middle and highest dose levels. Stillborn pups were detected in one mother from the highest dose level (all litter). Effect of cannibalism of pups in female was observed in the lowest and highest doses as well as in the control group.

Male and female fertility index and was decreased in the middle and highest dose levels. The gestation index was decreased in females at the lowest and highest dose levels. The post-natal losses were not significantly changed in treated and control groups. Markedly increased post-implantation losses in females in the highest dose level were recorded.

The total number of pups was decreased in females in all treated groups, but in the highest dose level decreased total number of pups was marked and with statistical and biological significance.

The decreasing of total number of pups and increasing of post-implantation losses were marked in the highest dose level and can be considered as biologically significant.

The weight of litters was statistically significantly decreased in highest dose level at all examination intervals and in the lowest and middle dose levels at the end of lactation period. The weight of pups was not changed compared to control group. No presence of nipples in male pups was recorded and anogenital distance in treated male and female pups was not biologically significantly affected. No macroscopical findings in pups were found out during pathological examination.

Accordingly, the NOAEL for reproduction was established as 250 mg/kg body weight/day. The NOAEL was established on the based marked increased post-implantation losses and marked decreased total number of pups in females in the highest dose level.

While, the NOAEL for development was established as 750 mg/kg body weight/day, indeed all changes in developmental parameters of pups observed at all dose levels were considered to be of no toxicological significance.

A further Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening is available on OB 2-DSA and is used within a weight of evidence approach.

The test item was doses to 150, 300 and 600 mg/kg/bw to rats and no toxicologically adverse effects on growth of animals, food consumption and health condition of animals were detected in both sexes.

The test item administration did not affect male ability to produce sperm that can fertilise eggs and ability of females to become pregnant.  

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in males. Decreased concentration of T4 hormone in males at the dose level 150 mg/kg/day was considered toxicologically insignificant.

The body weight of parental males and females was not significantly affected by the test item application.

Evaluation of the absolute and relative weight of reproductive organs of male and female, as well as the weight of pituitary and thyroid gland revealed only sporadic findings. The absolute weight of testes and epididymis was significantly decreased in males at the dose level 600 mg/kg/day (also reduced absolute weight of testes was noted in males at the dose level 150 mg/kg/day). Evaluation of relative weight of reproductive organs in males, absolute and relative weights of reproductive organs in treated females did not reveal any statistically significant differences compared to control.

Histological examination did not reveal negative effect of the test item on collected reproductive organs, pituitary and thyroid glands.

Spermatogenesis in the testes of the high dose administered males was without any pathological findings. Epididymides of both control and high dose males were without any pathological findings. Sporadic findings were of spontaneous origin.

Examination of sperm motility and morphology in treated parental males did not show any differences in comparison with the control males.

The number of implantations was comparable between treated and control groups.

The calculated parameters (mating and fertility indexes) were similar in all treated groups in comparison with the control groups. Fertility indexes were slightly lower in high dosed groups in comparison with the control group due to one non-pregnant female. Gestation indexes in treated groups were identical with the control group. The viability index was the best in the highest dosed group of females.

The test item did not affect the number of pups and their development.

The total number of pups, mean number of pups per litter and mean litter weight at first litter check after parturition and during the next intervals were similar or higher in treated groups of mothers compared to control group. The mean pup body weights at all checking intervals was slightly lower in all treated groups compared to control group due to higher number of pups per group of treated females. Macroscopical examination of pups did not show negative effect of the test item administration on development of pups.  

No adverse effect of the test item treatment was observed during examination of thyroxine and rat thyroid stimulating hormone blood concentration in pups. Male nipple retention at day 13 was not recorded in any male pup. Corrected anogenital distance in treated pups was comparable to the control pups.  

Based on these findings, the NOAEL value for reproduction and development was established as 600 mg/kg/day since no biologically significant changes relevant to reproduction and development were observed.

Overall, no toxicity for the development of foetuses was observed in none of the studies above described. Therefore, considering the absence of significant adverse effects on developmental parameters even in the case of disulfonated aniline derivates, it seems reasonable to complete the assessment of this toxicological potential using the data available on the less polar substances belonging to the same category in a weight of evidence approach.

Firstly, this toxicological potential was assessed using the OECD 414 test on 1-MSA (report n. 20-49, 2020).

In particular, the study was conducted by oral gavage as administration route; this was achieved by removing the access to the diet 2 hours prior to the gavage administration and by giving access to the diet 2 hours after the administration, in order to limit the possibility of association to food components for the tested substance.

A dose-range finding experiment (DRFE) was performed to determine the dose levels for the main study. In this test, oral administration of the test item to pregnant females by gavage from the 5th to the 19th day of pregnancy at the dose levels 100, 300 and 1000 mg/kg/day did not cause mortality of pregnant females.

No adverse changes of health conditions and no clinical symptoms of intoxication were found in females at any dose level after administration of the test item.

The negative effect of the test item on the growth of maternal animals was not observed at any dose levels.

Haematological examination did not show significant differences among the dose levels. Pathological examination of females and foetuses revealed no serious macroscopic changes.

There were changed reproduction parameters at the dose level 1000 mg/kg/day, i.e. decreased number of implantation and decreased number of foetuses. Based on these findings and on all available data for other category’s member (following described), the highest tested dose for the main study was selected as follows: 0, 100, 300 and 750 mg/kg/day.

After acclimatization and mating, the test item was administered to pregnant females by gavage, daily from the 5th to the 19th day of pregnancy.

Under test conditions, no negative effects on the growth of maternal animals were observed.

Clinical and pathological examinations of treated mothers did not detect symptoms of toxicity related to the test item treatment.

Reproductive parameters (implantations, corpora lutea, pre- and post-implantation losses) were unaffected by treatment with the test item.

Examination of the thyroid gland (absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones) did not reveal any changes associated with the application of the test item.

One dead foetus was only found at the highest dose level. The average total number of live foetuses in litter was well balanced in all groups.

Based on statistical evaluation of mean values of foetal body weight, no significant growth retardation was detected in treated groups. The foetal body weight was non-significantly slightly decreased at the highest dose level, although litter size was comparable in all groups.

The mean anogenital distance and corrected AGD of male foetuses at the all dose levels were balanced with control. As for females, the mean anogenital distances was statistically significantly increased only at the middle dose level; while the mean corrected anogenital distance of female foetuses was comparable at all dose levels with control group.

The test item treatment did not evoke occurrence of external and visceral variations and malformations.

Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded in foetuses of all treated groups as well as control. It is normal variability in the schedule of ossification; ossification of sternum have not to be complete on the 20th day of pregnancy. These findings were not related to the treatment, because the occurence of these findings was comparable or lower to the control group.

Examination of vertebrae revealed high incidence of dumbbell ossification of vertebrae thoracic centrum in all test groups including control group. There was recorded increased dose-dependent incidence of litters with dumbbell ossification of vertebrae thoracic centrum in the treated groups compared to the control group (76.19 %–79.17 %–85.00 %–100.00 %) but the incidence of affected foetuses with this finding at treated groups was quite comparable with control group (33.33 % – 33.16 % – 31.25 % – 40.63 %), highlighting no dose-dependency of the effect. The statistical significance was not detected. The interpretation of this finding is questionable, due to the high incidence of this finding also in the control foetuses. The dumbell ossification of vertebrae thoracic centrum is included in the Transitional Findings (grey zone). These may be upgraded to malformation or downgraded to variations status, depending on severity and/or frequency of occurrence.

The changes such as wavy ribs and ribs-supernumerary site were detected also in all groups. The portions of litters with wavy ribs and ribs-supernumerary site were similar or lower at all dose levels compared to the control group. These findings did not relate with test item treatment.

The incidence of incomplete ossification of scapula was recorded only in litters at treated groups, without dose relationship and statistical significance. This variation is not considered to be adverse.

The NOAEL for toxicity in PREGNANT FEMALES was established as 750 mg/kg/day. This NOAEL value is based on no mortality of females, no changes in health condition status, no pathological findings in the dams, no dose related changes in reproduction parameters up to this dose level.

The NOAEL for PRENATAL DEVELOPMENT was established as 750mg/kg/day. The occurrence of the dumbbell ossification of vertebrae thoracic centrum in all litters at the dose level 750 mg/kg/day was not considered as adverse due to the high incidence in control litters and because the incidence of affected foetuses with this finding at treated groups was quite comparable with control group.

Within the category, two other tests were carried out on Wistar CRL rats according to the OECD guideline 414 and under GLP conditions on similar substances OB 2-A and OB 5-A (report n. 19-489, 2019 and 86/19/11, 2020), using conditions and doses equal to the ones applied for the target substance. They gave comparable results and were used in weight of evidence within a read-across approach.  

With regard to OB 2A, under test conditions, there were no unscheduled deaths, no adverse changes of health condition and no clinical symptoms of intoxication in females at any dose level after administration of the test item.

Macroscopical structure of organs of pregnant females and values of reproduction parameters (number of females with live foetuses, number of live and dead foetuses, early and late resorptions and sex ratio of foetuses) were unaffected by treatment with the test item.

Examination of the thyroid glands, in terms of absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones, did not reveal any changes associated with the application of the test item. Only serum level of TSH was slightly decreased in females at the middle dose level, but without statistical significance.

Test item-related foetal mortality was not evident at any dose level. The foetal body weight was statistically insignificantly decreased at the middle dose level. The mean anogenital distance of male foetuses was statistically significantly increased at the lowest and highest dose levels compared to the control. The corrected male AGD was statistically significantly increased only at the lowest dose level. In male foetuses we did not observe the feminization effect (shortening male AGD). The mean AGD and corrected AGD of female foetuses at the dose levels was balanced with control.

Detailed necropsy of foetuses did not reveal increase of external and visceral variations and malformations at any dose level.

Foetal skeletal examination revealed no statistically significant differences between treatment groups and control group.

The NOAEL for toxicity in pregnant females was established as 750 mg/kg/day.

The NOAEL for prenatal developmental was established as 750 mg/kg/day.

Even, 5A had no negative effect on the growth of maternal animals.

Clinical and pathological examinations of treated mothers did not detect signs of toxicity related to the test item treatment. The behavior, health condition and clinical status of treated maternal animals were similar compared to the control and no serious changes were found.

Pathological examination of females revealed no pathological finding related to the test item treatment. Evaluation of the uterine weights did not demonstrate a negative effect of the test item treatment too.

The examinations of reproductive parameters (number of live foetuses, early and late intra uterine death, numbers of implantations, corpora lutea and resorptions) in all treated groups were not affected by the test item treatment. Post-implantation losses (IUDL) were increased at the lowest dose level in comparison with control group, but without statistical and toxicological significance.

Examination of the thyroid gland– absolute and relative weight of thyroid gland, histological examination of thyroid gland and serum levels of thyroid hormones, did not reveal any changes associated with the application of the test item.

Two dead foetuses were only found at the middle dose level. The average total number of live foetuses in litter was well balanced in all groups.

Based on statistical evaluation of mean values of foetal body weight, no significant growth retardation was detected in treated groups. The foetal body weight was slightly decreased at the lowest dose level, without statistical and toxicological significance.

The mean anogenital distance and corrected AGD of male and female foetuses at the all dose levels were balanced with control and not affected with the test item application.

The test item treatment did not evoke occurrence of external and visceral variations and malformations connected with the test item treatment. A deformity of the end part of the spine, short hind-limbs and absent tail were found out only in one female foetus at the lowest dose level. These findings observed in this one foetus were not treatment related and were of spontaneous origin.

Examination of foetal skeleton indicated mainly delayed development of the skeleton at all dose levels as well as in the control group.

Incomplete ossification of foetal cranium was found out during examination in all groups including control group. With delayed development were affected mostly parietal bone, interparietal bone, supraoccipital bone and squamous part of temporal bone, less frequently frontal bone, arcus zygomaticus and basisphenoid. This delayed development was not related to the treatment, due to a similar percentage in dosed groups and control group. Only the portions of litters with incomplete ossification of basisphenoid (15.79 %–10.53 %– 21.05%– 21.74 %) and arcus zygomaticus (10.53 % – 10.53 % – 15.79 % – 17.39 %) were very slightly increased at the middle and highest dose levels, without statistical and toxicological significance. The incidence of incomplete ossification of supraoccipital bone in foetuses at the lowest dose level was statistically significantly increased compared to the incidence in control foetuses (76 – 124 – 81 – 116 foetuses), without dose dependence and toxicological significance. This finding was not related to the treatment, because the occurence of this finding in litters was high in all dose levels and also in control group (89.47 % – 100.00 % – 94.74 % – 95.65 %) and dose dependence was not evident. Other findings on cranial bones, which were observed in all test groups including control group, was hole in the supraoccipital bone. The frequency of holes in the supraoccipital bone was lower at the dose levels compared to the control group.

Incomplete ossification of ossification sites of sternebra and unossified ossification sites of sternebra were recorded in foetuses of all treated groups as well as control. It is normal variability in the schedule of ossification, ossification of sternum have not to be complete on the 20thday of pregnancy. These findings were not related to the treatment, because the occurrence of these findings was comparable or lower to the control.

Examination of vertebrae revealed dumbbell and bipartite ossification of vertebrae thoracic centrum in all test groups including control group.

The changes, such as wavy ribs and ribs-supernumerary site, were detected also in all groups. The portions of litters with wavy ribs were very slightly increased at the middle and highest dose levels (15.79 % – 5.26 % – 21.05 % – 21.74 %) compared to the control group, without statistical and toxicological significance. The incidence of litters with the ribs supernumerary site was increased at the middle dose level (36.84 %–10.53 %–63.16 %–39.13 %), without dose relationship.

The NOAEL for toxicity in PREGNANT FEMALES was established as 750 mg/kg/day.

The NOAEL for PRENATAL DEVELOPMENT was established as 750 mg/kg/day.

Another study on developmental toxicity and teratogenicity is available for a further similar substance (OB 3a-MSA). The study was conducted according to EPA Guideline OPPTS 870.3700, under GLP conditions (report n. 795-003, 1999).

Under test conditions, 30 pregnant Sprague-Dawley rats per group were dosed with 100, 400 or 1000 mg/kg bw/day by oral gavage on gestation days 6-19.

The only substance-related effect observed was discoloured faeces at 400 and 1000 mg/kg bw/day. At skeletal examination of foetuses, the incidence of misaligned sternebra was slightly increased in all dose groups but was well within historical control range and not dose-related and therefore not considered to be test substance related. The incidence of rudimentary ribs was slightly above the historical control range at 100 and 1000 mg/kg bw/day. As the difference from the concurrent control group was not statistically significant and the increase was not dose-related, these findings were not considered biologically significant or test substance-related.

The number of vertebral malformations at 1000 mg/kg bw/day (litter incidence 7.1 %) was very slightly above the historical control range (0 - 7 %) and not statistically different from the vehicle controls. Therefore, also this border finding was considered to be within normal variation and unrelated to test substance administration.

As there were no adverse maternal or developmental effects seen at any dose level, the NOAEL for both maternal and foetal toxicity is the highest dose tested (1000 mg/kg bw/day).

A further pilot prenatal developmental toxicity study was performed in rats with OB 2-A free acid, administered via oral gavage. 7 groups each of 10 mated female Sprague-Dawley rats per group were treated once per day via oral gavage either with the vehicle alone (one control group) at the dose levels of 30, 300, or 1000 mg/kg bw/day. Dosing was initiated on Day 6 of gestation and continued to and included Day 19 of gestation. Clinical signs, gestational body weight, and food consumption were recorded. Litters were delivered by laparohysterectomy on Day 20 of gestation. Gravid uterine weights were recorded. Total number of corpora lutea, implantations, early and late resorptions, and live and dad fetuses were recorded.

No adverse treatment-related maternal or developmental effects were observed at any dose level.

NOAEL maternal and developmental: 1000 mg/kg bw/day.

In conclusion, the NOAEL values found in all the available studies are consistent, confirming the absence of concern for developmental and maternal toxicity in rats regarding this category of substances.

In this respect, the value from the study carried out on OB 2-DSA, according to OECD TG 422, where the NOAEL was established to be 600 mg/kg bw/day, was selected as representative for the disulfonated aniline derivates.

Regarding the second species, a developmental toxicity study on rabbits, carried out according to the EPA guidance 870.3700 and under GLP conditions, is available for OB 3a-MSA and rises some concern for maternal toxicity (report n. 795-004, 2000).

The substance was tested in rabbits in accordance with OPPTS Guideline 870.3700, EPA Guidelines. The dose levels of 100, 400, and 800 mg/kg/day were administered via gavage with 0.5 % CMC (carboxymethyl cellulose) in water to 3 treatment groups of 25 females and 1 vehicle control group.

Exposure initiated on Day 7 of gestation and continued to and included Day 28 of gestation.

Excessive maternal toxicity, as evidenced by mortality, statistically significant decreases in body weight gain and food consumption as well as an increase in abortion, was observed at 800 mg/kg/day. As a result, this group was terminated prior to completion of the study. Less severe maternal toxicity was observed at 400 mg/kg/day.

Two does died in the control group, but these deaths were a result of technical gavage error or mechanical injury. A total of 8 rabbits from the 800 mg/kg/day group died during gestation and another high-dose doe was euthanized in extremis. 7 does from the high-dose group aborted during the study. No treatment-related mortality was noted at 100 or 400 mg/kg/day.

Treatment-related clinical observations at 400 mg/kg/day included soft faeces and discoloured stool. No changes in body weight, body weight gain or food consumption were noted at 100 or 400 mg/kg/day.

Necropsy findings in does from the 800 mg/kg/day group included discoloration of the liver, edematous and/or discoloured stomach, red discoloured and/or edematous intestines, bloody and/or mucoid contents in the intestines. The rabbit in the 400 mg/kg/day group that aborted had an edematous stomach and liquid, bloody contents in the intestines at necropsy which were also considered to be treatment related.

No effects on uterine parameters were noted at 100 or 400 mg/kg/day.

Numbers of corpora lutea, implantations, live and dead foetuses and resorptions were comparable between the vehicle control and the 100 and 400 mg/kg/day groups.

Foetal body weights were statistically lower at 400 mg/kg/day when compared with the vehicle control group.

Based on treatment-related clinical observations and necropsy findings seen in does at 400 mg/kg/day, the No Observed Adverse Effect Level (NOAEL) for maternal effects in this study was 100 mg/kg/day, the lowest dose tested. There were statistically significant decreases in foetal body weight at 400 mg/kg/day. These changes may have been secondary to the maternal toxicity observed in this study and were not considered to be an indication of developmental toxicity.

The NOAEL value for developmental toxicity was established to be 400 mg/kg/day.

The study results did not indicate concern for the rabbit prenatal developmental toxicity, but evidenced a higher toxicity of the test substance in rabbits in comparison with rats.

However, pre-natal developmental toxicity studies are not designed to investigate species dependent toxicity for adult animals.

Due to the possibility of the substances to associate to food components, this NOAEL value could be not representative of the real toxicity of these substances. Accordingly, to consolidate the category approach with additional experimental information, new developmental toxicity studies in rabbits were planned on OB 2-A and on OB 4-MSA. In addition, the studies were designed to minimize contact of the test material with the constituents of the standard diet used in animal testing, investigating the extent of such association for this class of compound and assessing the toxicity with food deprivation.

It was decided to start the new studies OECD TG 414 in the rabbit testing the compound OB 4-MSA, which has a 2-hydroxypropyl amino function in R2 not otherwise present in the constituents of the SFWA substances. Furthermore, OB 4-MSA has the highest content of non-specified impurities.

Unfortunately, this study was stopped at the dose range finding experiment due to methodological limitations. The test item was administered by oral gavage from Day 6 to Day 28 of gestation at the dose levels of 100, 300 and 750 mg/kg/day to 6 animals per dose (report n. A4103, 2020). Food was removed each day of dosing from 3 hours before and up to 3 hours after administration (total of 6 hours of fasting). Control group animals received softened water and they were also subjected to the resection of food like the treated animals. In order to demonstrate the emptying of the stomach after 6 hours of fasting, three animals of the control group and three animals of the high dose group were sacrificed (interim sacrifice) before gestation Day 29 and the full and empty stomach were weighed.

During the in-life phase of the study, 3 high dose animals were sacrificed on gestation Day 17 or 19 and one mid-dose female was sacrificed on gestation Day 25. All animals were sacrificed due to the presence of red staining or foetuses on the cage tray.

Clinical signs and maternal necropsy of animals that reached term revealed no treatment related changes. The interim sacrifice of three control and three high dose rabbits killed after the completion of the 6 hours fasting period demonstrated that is not sufficient to completely empty the stomach.

At the highest dose tested, i.e. 750 mg/kg bw/day, two rabbits were sacrificed on gestation Day 17 and one rabbit aborted on gestation Day 19. Two rabbits lost body weight (animals that showed foetal total resorption at necropsy) on gestation Day 15 and showed reduced faeces from Day 13 or 14 of gestation.

Reduced faeces is indicative of reduced food intake consumption or reduced intestinal motility. The remaining rabbits, which aborted on gestation Day 19 did not show any clinical signs. Body weight change (%) in animals was lower than controls from Day 9 of gestation. At necropsy, all three rabbits showed changes at the gastrointestinal tract: stomach or caecum was found thickness in the two rabbits sacrificed during the mid- gestation and brown fluid was found the caecum of rabbit, which aborted on gestation Day 15.

One mid-dose rabbit aborted on Gestation Day 25. The animal did not show any clinical signs and no maternal abnormalities were found at necropsy. This occurrence of abortion is unclear, but considering that rabbits are very sensitive to stress caused by food deprivation or food restriction, it may be a treatment related effect exacerbated by the not ad libitum feeding. Body weight change (%) in animals was lower than controls from Day 9 of gestation. No treatment related effects were observed in clinical signs, including maternal reproductive parameters, mean numbers of corpora lutea, implantation sites, live and dead foetuses, litter size, and pre and post implantation losses in animals sacrificed on gestation Day 29. There were no treatment related effects on foetal sex ratio or survival at term. No treatment related effects on foetal development were seen.

Body weight change (%) in animals was slightly lower than controls from Day 9 of gestation.

With regard the low dose group, no treatment related effects were observed on body weight and clinical signs, including maternal reproductive parameters, mean numbers of corpora lutea, implantation sites, live and dead foetuses, litter size, and pre and post implantation losses. There were no treatment related effects on foetal sex ratio or survival at term. No treatment related effects on foetal development were seen.

On the basis of the results, it can be concluded that the test item at the dose level of 750 mg/kg/day is not tolerated by pregnant rabbits, as demonstrated by the clinical signs and the changes noted at the gastrointestinal tract at necropsy. As a consequence of the gastrointestinal disturbance the animals aborted or were sacrificed for humane reasons. The interim sacrifice of three control and three high dose rabbits killed after the completion of the 6 hours fasting period demonstrated that is not sufficient to completely empty the stomach.

All the above issues may be attributable or enhanced by the stress generated by food deprivation.

Indeed, it should be noted that rabbits are very sensitive to stress and one of the factors that induce stress is food deprivation or food restriction. The effects of stress can be potentially lethal for rabbits.

The stress may affect the toxicological evaluation of the study resulting in incorrect conclusions for human safety assessment. Although there was no mortality in the control group, the stress (induced in mothers by food restriction) affected adversely the foetal development. The number of small foetuses (foetal weigh less than 35 g) in the control group is much higher than our historical data and the mean foetal weight is lower than historical control data (-14%).

Therefore, this study was unable to shade light on the concern arisen from the developmental study on rabbits with OB 3a-MSA.

In conclusion, no toxicity for the development of foetuses was observed in none of the studies above described, included the one conducted on rabbits and the Dose Range Finding study on rabbit.

However, the available studies demonstrate likely differences in toxicity profile between rat and rabbit. The non-rodent species seems to be more sensitive to the treatment with the SFWA category in terms of maternal toxicity.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.7 Reproductive toxicity section, reproductive toxicity includes adverse effects on sexual function and fertility in adult males and females, as well as developmental toxicity in the offspring.

The available experimental data are adequate for classification and labelling and the substance is not classified for reproductive and developmental toxicity according to the CLP Regulation (EC 1272/2008).