Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C9 odd-numbered alkyl) derivs. and sodium hydroxide and chloroacetic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed with an analogue. The results are read across to the registered substance following the rationale attached in Section 13. Based on the results, the parental and developmental NOAEL were found to be at least 300 mg/kg bw/day. The NOAEL for reproduction was at least 1000 mg/kg bw/day.

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:

The rationale to read across the data is attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Vehicle:

water

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
Salivation occurred in most males in the 1000 mg/kg bw/day dose group throughout the treatment period. One male showed flat posture, red discoloration on the nose and laboured respiration and rales shortly before early sacrifice on day 20. The clinical signs among the surviving males comprised occasionally laboured breathing, rales, piloerection and red discolouration or discharge on the nose, generally during the reproductive period only. Salivation occurred from time to time during the premating and reproductive periods in several males in the 300 mg/kg bw/day dose group. Single males had severe rales, piloerection and a red discoloration on the nose sporadically throughout the treatment periods (premating and reproductive). In the 100 mg/kg bw/day dose group, piloerection, a scab on the tail and salivation occurred in single males in this dose group only on the first day one of dosing (salivation) or during the reproductive period (piloerection, scab).

Females
Salivation occurred in most females in the 1000 mg/kg bw/day dose group throughout the treatment period. Four females died prematurely (see "Mortality"). The clinical signs among the surviving females comprised occasional rales and piloerection during the treatment period. Salivation occurred from time to time during the premating and reproductive periods (post coitum, lactation) in some females in the 300 mg/kg bw/day dose group. Single females had hunched posture and laboured respiration once during the study. One to two females had piloerection, alopecia, scabs (cervical region) and a wound (cervical region) during the reproductive period.
In the 100 mg/kg bw/day dose group, salivation occurred sporadically in one or two females during the treatment period.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were five animals (one male and four females) treated at 1000 mg/kg bw/day that were found death or sacrificed in extremis.
The male was sacrificed in extremis on study day 20. Prior to euthanasia, this male had a flat posture, red discoloration on the nose and laboured respiration, rales and salivation. It gained weight prior to death. There were no relevant macroscopic findings but microscopic findings included slight necrosis of the tracheal
epithelial, which may be suggestive for a gavage-related procedural incident. However, it cannot be excluded that like in the females discussed below, the morbidity may have been caused by regurgitation secondary to the test item. All other males survived to scheduled euthanasia.

Three females treated at 1000 mg/kg bw/day were prematurely euthanized, due to respiratory clinical signs and one was found dead (3 females before mating at day 12-16 and 1 female at day 26). One female was euthanized in extremis on study day 12. Prior to euthanasia this female had slight hunched posture, severe laboured respiration and rales, piloerection, moderate salivation and was severely pale. The female lost weight prior to death.
One female was found dead on study day 15. This female showed rales on day 1 and salivation during treatment, but no other clinical signs prior to death.
One female was euthanized in extremis on study day 26. Prior to euthanasia this female had piloerection and severe laboured respiration, rales, salivation and ptosis. One female was euthanized in extremis on study day 16. Prior to euthanasia this female had slight quick breathing, piloerection and severe rales, shallow respiration, salivation and ptosis. The female lost weight prior to death. Macroscopically the lungs were not collapsed which microscopically could be explained by trachea and lung lesions (up to marked degree) such as: acute inflammation (trachea and lung), ulceration/erosions of bronchial epithelium and trachea, and bronchial fibrosis and/or hyperplasia. In one female, foreign material was found within the tracheal lumen.

Description (incidence and severity):

Body weights and body weight gain of treated males remained in the same range as controls over the treatment period in the 100 and 300 mg/kg bw/day dose groups. In the 1000 mg/kg bw/day dose group male body weights and body weight gain were reduced, achieving levels of statistical significance on Day 15 and 29 of treatment (Day 1 and 15 of mating, respectively), resulting in a 9% lower mean body weight at the end of treatment compared to concurrent controls. It was noted that one male at 1000 mg/kg bw/day lost weight over the first week of treatment, but recovered during the second week. In the absence of any clinical sign during the first week, no toxicological significance was attached to this finding.

Body weights and body weight gain of treated females remained in the same range as controls over the treatment period in the 100 and 300 mg/kg bw/day dose groups and in the 1000 mg/kg dose group until the early termination.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for the males and females. A low food consumption was recorded for one cage 8, containing five high dose males, over the first week of treatment. One of the males in this cage had lost weight during this period and it was considered likely that this was accompanied by a lower food consumption which consequently affected the food consumption value for this cage.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg bw/day dose group males, a decreased number of reticulocytes was observed, achieving a level of statistical significance when compared to controls. Furthermore, a slightly higher mean corpuscular haemoglobin concentration (MCHC) value was calculated from the red blood cell parameters, also achieving a level of statistical significance when compared to the MCHC value in controls. No toxicologically relevant changes were noted in the other haematological parameters in males at 1000 mg/kg bw/day and in all haematology parameters in males at 100 and 300 mg/kg bw/day.
The haematology results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice) and those for the females of the other groups (including controls) for PND 14-16 (at lactation). The slightly increased values for red blood cell, reticulocyte and platelet counts, and corresponding changes in Red Blood Cell Distribution Width and red blood cell derived indices Mean corpuscular haemoglobin and Mean corpuscular volume, in 1000 mg/kg bw/day dose group females in comparison with controls, were likely due to the difference in their physiological status, rather than indicative of a treatment-related effect.
Although historical control data representative for Day 14 post-coitum were not available, the results obtained in females at 1000 mg/kg bw/day in this study were all within the normal range and it was concluded that there were no (marked) changes in any of the haematology parameters that indicated a treatment-related effect at sacrifice on Day 14 postcoitum. No toxicologically relevant changes were noted in haematological parameters in females treated at 100 and 300 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Coagulation parameters of treated males and female rats were considered not to have been affected by treatment.
Unexpected high mean values for Prothrombin Time and Activated Partial Thromboplastin Time were observed in control males in comparison with the historical control data. Since the mean values in treated males were similar to the historical control data and they showed no dose-response relationship, no toxicological significance was attached to the statistical significances apparent for PT in the treated groups.
No toxicologically relevant changes were noted in clinical biochemistry parameters. Statistically significant reductions in Aspartate aminotransferase in the 100 and 300 mg/kg bw/day dose groups were considered the have occurred by chance. Since no dose-response was present and a decrease in this enzyme level in plasma is of no biological relevance, no toxicological significance was attached to this finding.
The clinical biochemistry results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice after 4 weeks of treatment) and those for the females of the other groups (including controls) for PND 14-16 (at lactation, after 7-8 weeks of treatment in this type of studies).
In the six early sacrificed females at 1000 mg/kg bw/day, the mean values for several parameters showed a statistically significant difference when compared to controls, comprising; Alanine aminotransferase, Alkaline Phosphatase, total protein, albumin, urea, cholesterol, potassium, calcium and inorganic phosphate. Although the available historical control ranges for the blood-value of these parameters were applicable for lactating females, the absolute blood-values in the early sacrificed, pregnant females at 1000 mg/kg bw/day were still within the historical control range of the laboratory. Therefore the changes were considered minimal and likely due to the difference in their physiological status, rather than indicative of a treatment-related effect. This assumption might also be supported by the fact that no treatment-related changes in the clinical biochemistry parameters were observed in males at 100 mg/kg bw/day after a similar treatment period of 4 weeks. In lactating females at 300 mg/kg bw/day statistically significantly lower levels for total protein and albumin were observed on PND 14-16. The mean values for these parameters were at the lower limit of the normal range of this laboratory. No treatment-related changes were observed in the other clinical biochemistry parameters in females at 300 mg/kg bw/day and in all clinical biochemistry parameters in females at 100 mg/kg bw/day.
Serum levels of T4 in F0 males were considered not to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Functional observation parameters were considered not to be affected by treatment in the males up to 1000 mg/kg bw/day and in the females up to 300 mg/kg bw/day. No functional tests were
performed in females at 1000 mg/kg bw/day, because of their early sacrifice. A large variation in motor activity, i.e. mean total movements and ambulations, was observed between the males the four dose groups. In the absence of a clear dose response relationship and since all activity was within the normal range the differences between groups were considered not indicative of a relation to treatment.
In females, the motor activity was similar between treated and control groups. No motor activity test was performed in females at 1000 mg/kg bw/day, because of their early sacrifice.
Moreover, males and females of all tested groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations in females up to 1000 mg/kg bw/day. Test item-related microscopic findings after treatment with Dehyton® DC were limited to the thyroid gland of the 300 and 1000 mg/kg bw/day group males: An increased incidence and severity in follicular cell hypertrophy in the thyroid gland of males was recorded at 300 mg/kg bw/day group (3/5 at minimal degree and 1/5 at slight degree compared to 1/5 at minimal degree in the controls) and at 1000 mg/kg bw/day group (2/5 at minimal degree and 2/5 males at slight degree, compared to 1/5 at minimal degree.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not to have been affected by treatment. All females, except one 300 mg/kg bw/day dose group female, had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 300 mg/kg bw/day (with normal litter). Given the incidental nature, absence of a dose-related incidence and absence of an apparent correlation to pregnancy status, this finding did not indicate a relation with treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.

Reproductive performance:

no effects observed

Description (incidence and severity):

There was 1/10 couples of the controls, 2/10 couples treated at 100 mg/kg bw/day and 1/10 couples treated at 300 mg/kg bw/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this.
In all females treated at 1000 mg/kg bw/day that were euthanized at Day 27-28, early placental/fetal development sites (development around day 9-12) were observed.
Mating index was considered not to be affected by treatment. Precoital time was considered not to be affected by treatment. All females showed evidence of mating within 5 days. Number of implantation sites was considered not to be affected by treatment.
Fertility index was considered not to be affected by treatment. A fertility index of 90%, 80%, 100% and 100% was observed for the controls and 100, 300 and 1000 mg/kg bw/day treated females respectively. It should be noted that a fertility index could be calculated for only 7/10 females at 1000 mg/kg bw/day. Three females of this dose group were dead before mating could have occurred.

Key result

Dose descriptor:

NOAEL

Remarks:

Parental

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: based on mortality due to regurgitation of the formulations

Key result

Dose descriptor:

NOAEL

Remarks:

Reproduction

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effecst seen up to and including the highest dose tested (1000 mg/kg bw/day)

Key result

Critical effects observed:

no

Neuropathological findings:

not examined

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment. Eight of 13 pups in one litter in the 300 mg/kg bw/day dose group had alopecia. As this finding was confined to a single litter it was considered not related to treatment. Alopecia is known to occur in pups and when it occurs within a litter is assumed to be genetic.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 1 after littering compared to the total number of offspring born was considered not to be affected by treatment.
The number of dead pups at first litter check was 0, 0, and 9 from three litters for the 0, 100, and 300 mg/kg bw/day dose groups, respectively, resulting in a live birth index of 100, 100 and 92%.
The low value in the 300 mg/kg dose group was considered to have occurred by chance, because 7 out of 9 dead pups belonged to one litter. A high litter-mortality
occasionally occurs, possibly indicating a possible lack of maternal care rather than any defect in the pups. One additional pup in this litter was missing on Day 2 postpartum. This pup had been noted as having less milk and a scab on its snout. Two additional pups in this litter that survived to planned necropsy also had a scab on the snout.

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. A viability index of 97%, 100% and 99% was observed for the controls and 100 and 300 mg/kg bw/day treated females, respectively.

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. A lactation index of 100%, 100% and 99% was observed for the controls and 100 and 300 mg/kg treated
females, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were considered not to be affected by treatment.

Sexual maturation:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment. Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment. Treatment up to 300 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Key result

Dose descriptor:

NOAEL

Remarks:

Development

Generation:

F1

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen at 300 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental and developmental NOAEL were found to be at least 300 mg/kg bw/day. The reproduction NOAEL was found to be at least 1000 mg/kg bw/day. These values are read across.

Executive summary:

A 28-day repeated dose toxicity study combined with a reproduction/developmental toxicity screening test was performed according to guidelines and GLP principles. Wistar Han rats were treated with Amphoacetates C8-C18 by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Males were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 50-54 days). Females that failed to deliver pups were treated for 41 days, with the exception of females in the 1000 mg/kg bw/day dose group that were euthanized or found dead (one female) at the end of the premating

period or during the post-coitum period due to adverse clinical observations. Accuracy and homogeneity of formulations determined by chemical analyses confirmed accurate dosing. At 1000 mg/kg bw/day, there was a high mortality in the females (4/10) and one premature

death in the males. These deaths were concluded to be related to regurgitation and thus secondary to the test item (possibly triggered by physical/chemical properties of the test-item solution in combination with the route of administration).

The surviving females in this group were early terminated shortly after the fourth female died, i.e. on Day 14 post-coitum. These early sacrificed Group 4 females, were all pregnant of a normal number of foetuses, and did not show any direct test item-related morphological changes.

There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis. No reproduction toxicity was observed up to the highest dose level tested (1000 mg/kg bw/day), although only 6/10 females at 1000 mg/kg could be examined. No developmental toxicity was observed up to the highest dose level evaluated (300 mg/kg bw/day) as none of the 1000 mg/kg bw/day dose group females were allowed to deliver due to either an early death or euthanasia based on adverse clinical observations.

Based on these results, the parental and developmental NOAEL were found to be at least 300 mg/kg bw/day. The reproduction NOAEL was found to be at least 1000 mg/kg bw/day. These values are read across.

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

Quality of whole database:

A reliable study is available (Klimisch 1), performed with an analogue.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

A combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test was performed with an analogue. The results are read across to the registered substance following the rationale attached in Section 13. Based on the results, the parental and developmental NOAEL were found to be at least 300 mg/kg bw/day.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

The rationale to read across the data is attached in Section 13.

Reason / purpose for cross-reference:

read-across source

Strain:

other: Crl: WI(Han)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation occurred in most females in the 1000 mg/kg bw/day dose group throughout the treatment period. Four females died prematurely (see "Mortality"). The clinical signs among the surviving females comprised occasional rales and piloerection during the treatment period. Salivation occurred from time to time during the premating and reproductive periods (post coitum, lactation) in some females in the 300 mg/kg bw/day dose group. Single females had hunched posture and laboured respiration once during the study. One to two females had piloerection, alopecia, scabs (cervical region) and a wound (cervical region) during the reproductive period.
In the 100 mg/kg bw/day dose group, salivation occurred sporadically in one or two females during the treatment period.

Mortality:

mortality observed, treatment-related

Description (incidence):

Four females treated at 1000 mg/kg bw/day were found death or sacrificed in extremis. The male was sacrificed in extremis on study day 20. Three females treated at 1000 mg/kg bw/day were prematurely euthanized, due to respiratory clinical signs and one was found dead (3 females before mating at day 12-16 and 1 female at day 26). One female was euthanized in extremis on study day 12. Prior to euthanasia this female had slight hunched posture, severe laboured respiration and rales, piloerection, moderate salivation and was severely pale. The female lost weight prior to death. One female was found dead on study day 15. This female showed rales on day 1 and salivation during treatment, but no other clinical signs prior to death. One female was euthanized in extremis on study day 26. Prior to euthanasia this female had piloerection and severe laboured respiration, rales, salivation and ptosis. One female was euthanized in
extremis on study day 16. Prior to euthanasia this female had slight quick breathing, piloerection and severe rales, shallow respiration, salivation and ptosis. The female lost weight prior to death.
Macroscopically the lungs were not collapsed which microscopically could be explained by trachea and lung lesions (up to marked degree) such as: acute inflammation (trachea and lung), ulceration/erosions of bronchial epithelium and trachea, and bronchial fibrosis and/or hyperplasia. In one female, foreign material was found within the tracheal lumen.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and body weight gain of treated females remained in the same range as controls over the treatment period in the 100 and 300 mg/kg bw/day dose groups and in the 1000 mg/kg bw/day dose group until the early termination.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically relevant changes in food consumption before or after correction for body weight were recorded for the females.

Water consumption and compound intake (if drinking water study):

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The haematology results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice) and those for the females of the other groups (including controls) for PND 14-16 (at lactation). The slightly increased values for red blood cell, reticulocyte and platelet counts, and corresponding changes in Red Blood Cell Distribution Width and red blood cell derived indices Mean corpuscular haemoglobin and Mean corpuscular volume, in 1000 mg/kg bw/day dose group females in comparison with controls, were likely due to the difference in their physiological status, rather than indicative of a treatment-related effect.
Although historical control data representative for Day 14 post-coitum were not available, the results obtained in females at 1000 mg/kg bw/day in this study were all within the normal range and it was concluded that there were no (marked) changes in any of the haematology parameters that indicated a treatment-related effect at sacrifice on Day 14 postcoitum. No toxicologically relevant changes were
noted in haematological parameters in females treated at 100 and 300 mg/kg bw/day.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Coagulation parameters of treated female rats were considered not to have been affected by treatment. The clinical biochemistry results in females should be interpreted with caution, because for the females at 1000 mg/kg bw/day the blood levels were representative for Day 14 post-coitum (the day of their early sacrifice after 4 weeks of treatment) and those for the females of the other groups (including controls) for PND 14-16 (at lactation, after 7-8 weeks of treatment in this type of studies). In the six early sacrificed females at 1000 mg/kg bw/day, the mean values for several parameters showed a statistically significant difference when compared to controls, comprising; Alanine aminotransferase, Alkaline Phosphatase, total protein, albumin, urea, cholesterol, potassium, calcium and inorganic phosphate. Although the available historical control ranges for the blood-value of these parameters were applicable for lactating females, the absolute blood-values in the early sacrificed, pregnant females at 1000 mg/kg bw/day were still within the historical control range of the laboratory. Therefore the changes were considered minimal and likely due to the difference in their physiological status, rather than indicative of a treatment-related effect. This assumption might also be supported by the fact that no treatment-related changes in the clinical biochemistry parameters were observed in males at 100 mg/kg bw/day after a similar treatment period of 4 weeks. In lactating females at 300 mg/kg bw/day statistically significantly lower levels for total protein and albumin were observed on PND 14-16. The mean values for these parameters were at the lower limit of the normal range of this laboratory. No treatment-related changes were observed in the other clinical biochemistry parameters in females at 300 mg/kg bw/day and in all clinical biochemistry parameters in females at 100 mg/kg bw/day.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No functional tests were performed in females at 1000 mg/kg bw/day, because of their early sacrifice. The motor activity was similar between treated and control groups. No motor activity test was performed in females at 1000 mg/kg bw/day, because of their early sacrifice. Moreover, males and females of all tested groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no test item-related alterations in organ weights.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The four females at 1000 mg/kg bw/day that were found dead or sacrificed in extremis, all showed non-collapsed lungs at necropsy. No other macroscopic abnormalities were found in these females. There were no macroscopic findings in any of the other female rats of all dose groups that were considered test item related.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations in females up to 1000 mg/kg bw/day.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was 1/10 couples of the controls, 2/10 couples treated at 100 mg/kg bw/day and 1/10 couples treated at 300 mg/kg bw/day that failed to deliver healthy pups. Histopathology did not reveal any changes in the reproductive organs that could explain this.
In all females treated at 1000 mg/kg bw/day that were euthanized at Day 27-28, early placental/fetal development sites (development around day 9-12) were observed.Fertility index was considered not to be affected by treatment. A fertility index of 90%, 80%, 100% and 100% was observed for the controls and 100, 300 and 1000 mg/kg treated females respectively. It should be noted that a fertility index could be calculated for only 7/10 females at 1000 mg/kg. Three females of this dose group were dead before mating could have occurred.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One female dosed at 300 mg/kg bw/day had implantations only. As this was not seen in the high dose group, this effects was considered to be incidental.

Early or late resorptions:

no effects observed

Description (incidence and severity):

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

The number of dead pups at first litter check was 0, 0, and 9 from three litters for the 0, 100, and 300 mg/kg bw/day dose groups, respectively, resulting in a live birth index of 100, 100 and 92%. The low value in the 300 mg/kg bw/day dose group was considered to have occurred by chance, because 7 out of 9 dead pups belonged to one litter. A high litter-mortality occasionally occurs, possibly indicating a possible lack of maternal care rather than any defect in the pups. One additional pup in this litter was missing on Day 2 postpartum. This pup had been noted as having less milk and a scab on its snout. Two additional pups in this litter that survived to planned necropsy also had a scab on the snout.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Gestation index and duration of gestation were considered not to be affected by treatment.

Changes in number of pregnant:

effects observed, non-treatment-related

Details on maternal toxic effects:

No signs of difficult or prolonged parturition were noted among the pregnant females. No deficiencies in maternal care were observed.

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: based on mortality due to regurgitation of the formulations

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Body weights of pups were considered not to be affected by treatment.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The number of live offspring on Day 4 before culling compared to the number of offspring on Day 1 was considered not affected by treatment. A viability index of 97%, 100% and 99% was observed for the controls and 100 and 300 mg/kg bw/day treated females, respectively.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was considered not to be affected by treatment.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The number of live offspring on Day 13 after littering compared to the number of live offspring on Day 4 (after culling) was considered not to be affected by treatment. A lactation index of 100%, 100% and 99% was observed for the controls and 100 and 300 mg/kg treated females, respectively.

External malformations:

no effects observed

Description (incidence and severity):

No macroscopic findings were noted among pups that were considered to be related to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen at 300 mg/kg bw/day

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

Based on the results of a combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the parental and developmental NOAEL were found to be at least 300 mg/kg bw/day. This result is read across.

Executive summary:

A 28-day repeated dose toxicity study combined with a reproduction/developmental toxicity screening test was performed according to guidelines and GLP principles. Wistar Han rats were treated with Amphoacetates C8-C18 by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 50-54 days). Females that failed to deliver pups were treated for 41 days, with the exception of females in the 1000 mg/kg bw/day dose group that were euthanized or found dead (one female) at the end of the premating period or during the post-coitum period due to adverse clinical observations. Accuracy and homogeneity of formulations determined by chemical analyses confirmed accurate dosing. At 1000 mg/kg bw/day, there was a high mortality in the females (4/10) and one premature death in the males. These deaths were concluded to be related to regurgitation and thus secondary to the test item (possibly triggered by physical/chemical properties of the test-item solution in combination with the route of administration).

The surviving females in this group were early terminated shortly after the fourth female died, i.e. on Day 14 post-coitum. These early sacrificed high dose females, were all pregnant of a normal number of foetuses, and did not show any direct test item-related morphological changes.

No developmental toxicity was observed up to the highest dose level evaluated (300 mg/kg bw/day) as none of the 1000 mg/kg bw/day dose group females were allowed to deliver due to either an early death or euthanasia based on adverse clinical observations. Based on these results, the parental and developmental NOAEL were found to be at least 300 mg/kg bw/day. This result is read across.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

A reliable study is available (Klimisch 1), performed with an analogue.

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

A 28-day repeated dose toxicity study combined with a reproduction/developmental toxicity screening test was performed according to guidelines and GLP principles. Wistar Han rats were treated with Amphoacetates C8-C18 by daily oral gavage at dose levels of 100, 300 and 1000 mg/kg bw/day. The rats of the control group received the vehicle, water, alone. Females that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and at least 14-16 days of lactation (for 50-54 days). Females that failed to deliver pups were treated for 41 days, with the exception of females in the 1000 mg/kg bw/day dose group that were euthanized or found dead (one female) at the end of the premating period or during the post-coitum period due to adverse clinical observations. Accuracy and homogeneity of formulations determined by chemical analyses confirmed accurate dosing. At 1000 mg/kg bw/day, there was a high mortality in the females (4/10) and one premature death in the males. These deaths were concluded to be related to regurgitation and thus secondary to the test item (possibly triggered by physical/chemical properties of the test-item solution in combination with the route of administration).

The surviving females in this group were early terminated shortly after the fourth female died, i.e. on Day 14 post-coitum. These early sacrificed high dose females, were all pregnant of a normal number of foetuses, and did not show any direct test item-related morphological changes.

No developmental toxicity was observed up to the highest dose level evaluated (300 mg/kg bw/day) as none of the 1000 mg/kg bw/day dose group females were allowed to deliver due to either an early death or euthanasia based on adverse clinical observations. Based on these results, the parental and developmental NOAEL were found to be at least 300 mg/kg bw/day.

Justification for classification or non-classification

Based on the current data-set, the registered substance is not classified for reproduction and developmental toxicity according to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures.