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Key value for chemical safety assessment

Effects on fertility

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:
The key study is GLP compliant and of high quality (Klimisch score = 1).
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 study was conducted to generate limited information concerning the effects of the test item (reaction product of amines, C12-14,-tert-alkyl, alcohol, C14-18, C18 unsat, and phosphorus pentoxide) on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition (Thorsrud, 2013). According to OECD Guideline 421, three treatment groups of twelve CD [Crl:CD(SD)] rats/sex/group were administered the test article at dose levels of 150, 500, or 1000 mg/kg/day. One additional group of twelve animals/sex served as the control and received the vehicle, peanut oil (arachis oil NF). The vehicle or test article was administered to all groups daily via oral gavage at a dose volume of 2 mL/kg/dose. Dosing began 14 days prior to pairing and continued to euthanasia (43 days total) for the parental (P) males. 250 observations of the P animals included clinical signs, body weights and body weight change, and food consumption during the premating/mating, gestation, and lactation periods, and parturition and litter data. Observations of the offspring (F1) included survival at birth and during lactation, individual clinical signs, pup body weights and sex, and gross abnormalities. At study termination, necropsy examinations were performed on all surviving P animals, and organs and tissues were collected, weighed, and examined for select groups. Dams losing their entire litter were subjected to a necropsy, and up to ten pups from the litters were saved in fixative for possible future examination. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.  

Once daily administration of "reaction product of amines, C12-14,-tert-alkyl, alcohol, C14-18, C18 unsat, and phosphorus pentoxide" by oral gavage to male rats for 7 weeks and female rats for two weeks prior to mating, through gestation and lactation at dose levels of 150, 500, and mg/kg/day produced clinical observations consisting of postdose salivation and yellow discolored hair in both male and female animals predominantly at 500 and 1000 mg/kg/day. Additional clinical findings were noted in the females predominantly at 500 and 1000 mg/kg/day that included increased activity, few/absent feces, and vulvar discharge. Two females at 1000 mg/kg/day had to be euthanized moribund close to the time of parturition. Test article-related changes in body weight and food consumption were limited to females at 500 and 1000 mg/kg/day. The F1 pup survival index and mean body weight were adversely affected by treatment with the test item at 500 and 1000 mg/kg/day.

Test article-related macroscopic findings were present in the adrenal gland, thymus, and stomach of females at 1000 mg/kg/day. Test article-related organ weight changes were present in the ovaries, liver, kidneys, adrenal glands, and thymus at 500 and 1000 mg/kg/day. Test article-related microscopic findings were present in the ovaries, liver, kidneys, adrenal glands, stomach, and duodenum. Most findings were predominantly observed at 1000 mg/kg/day, but were also noted to some extent in animals at 500 mg/kg/day.

Based on the results obtained from this study, the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity in parental male and female animals was considered to be 150 mg/kg/day, which was generally based on, but not limited to, significant clinical findings, decreases in body weight and food consumption (primarily females), as well are organ weight changes and microscopic findings at 500 and 1000 mg/kg/day. A No-Observed-Effect-Level (NOEL) for F1offspring for developmental toxicity was considered to be 150 mg/kg/day, based on the significant decreases in survival and mean body weight at 500 and 1000 mg/kg/day. However, mating, fertility, and fecundity indices were unaffected by treatment with the test item at dose levels up to 1000 mg/kg/day, the highest dose level tested.


Short description of key information:
No effects on fertility were reported. NOAEL of 1000 mg/kg bw was established for reproductive performance (Thorsrud, 2013; OECD 421).

Justification for selection of Effect on fertility via oral route:
Only one study available.

Justification for selection of Effect on fertility via inhalation route:
No relevant route of exposure.

Justification for selection of Effect on fertility via dermal route:
The substance is neither a skin irritant nor a skin sensitiser. Thus, the evaluation of this endpoint is not justified.

Effects on developmental toxicity

Description of key information
Based on decreased mean fetal body weights with an associated increase in the mean litter proportions of skeletal developmental variations related to reduced ossification at 1000 mg/kg/day, a dosage level of 500 mg/kg/day was considered to be the NOAEL for embryo/fetal development (Herberth, 2015; OECD 414). No direct developmental toxicity can be attributed to the test substance since fetal effects observed at 1000 mg/kg bw were occurred in the presence of maternal toxicity and the corresponding decrease in food consumption in the dams.
In the screening study, a No-Observed-Effect-Level (NOEL) for developmental toxicity was considered to be 150 mg/kg/day, based on the significant decreases in survival and mean body weight at 500 and 1000 mg/kg/day doses (Thorsrud, 2013; OECD 421). Similarly, developmental effects observed at 500 mg/kg bw and 1000 mg/kg bw were associated with maternal toxicity effects at these dose levels: NOAEL for general toxicity in parental male and female animals was considered to be 150 mg/kg/day, which was generally based on, but not limited to, significant clinical findings, decreases in body weight and food consumption (primarily females), as well are organ weight changes and microscopic findings at 500 and 1000 mg/kg/day.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP compliant and of high quality (Klimisch score = 1).
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

Prenatal developmental toxicity study (OECD 414)

The study was conducted to determine the potential of the test substance (reaction products of alcohols, C14-18, C18 unsat., esterified with phosphorus pentoxide and salted with amines, C12-14,-tert-alkyl) to induce developmental toxicity after maternal exposure from implantation to 1 day prior to expected parturition, to characterize maternal toxicity at the exposure levels tested, and to determine a no-observed-adverse-effect level (NOAEL) for maternal toxicity and developmental toxicity (Herberth, 2015).

The test substance, in the vehicle (arachis [peanut] oil) was administered orally by gavage to 4 groups of 25 bred female Crl:CD(SD) rats once daily from gestation days 6 through 19. Dosage levels were 100, 300, 500, and 1000 mg/kg/day administered at a dosage volume of 5 mL/kg. A concurrent control group composed of 25 bred females received the vehicle on a comparable regimen. The females were approximately 14 weeks of age at the initiation of dose administration. All animals were observed twice daily for mortality and moribundity. Clinical observations, body weights, and food consumption were recorded at appropriate intervals. On gestation day 20, a laparohysterectomy was performed on each female. The uteri, placentae, and ovaries were examined, and the numbers of fetuses, early and late resorptions, total implantations, and corpora lutea were recorded. Gravid uterine weights were recorded, and net body weights and net body weight changes were calculated. The fetuses were weighed, sexed, and examined for external, visceral, and skeletal malformations and developmental variations.

All females in the control, 100, 300, 500, and 1000 mg/kg/day groups survived to the scheduled necropsy on gestation day 20. Test substance-related incidences of red material around the nose and mouth, decreased defecation, pale feces, and yellow material around the urogenital area were noted for the 1000 mg/kg/day group at the daily examinations and/or approximately 1 hour following dose administration generally throughout the treatment period. Test substance-related, non-adverse increased incidences of clear material around the mouth was noted in all test substance-treated groups and red material around the nose was noted for the 100, 300, and 500 mg/kg/day groups at the daily examinations and/or approximately 1 hour following dose administration primarily during the first week of treatment (gestation days 7-13). No other test substance-related clinical findings were noted at the daily examinations or 1 hour following dose administration at any dosage level. However, at the daily examinations 3 females in the 1000 mg/kg/day group had hunched posture on gestation day 20 and 1 female in this group had clonic convulsions on gestation day 19. These observations were considered transient and not attributed to test substance administration.

Test substance-related mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption were noted in the 1000 mg/kg/day group compared to the control group throughout the treatment period. In addition, lower mean body weights, net body weight, and net body weight gains were observed at this dosage level.

In the 500 mg/kg/day group, a lower mean body weight gain with corresponding lower mean food consumption was noted compared to the control group during gestation days 6-9. Mean body weight gains and food consumption in this group were generally similar to the control group throughout the remainder of the treatment period. As a result of the initial body weight decrements, a lower mean body weight gain was noted for the 500 mg/kg/day group when compared to the control group for the overall treatment period (gestation days 6-20). However, the lower mean body weight gains noted for the 500 mg/kg/day group were not of sufficient magnitude to affect mean body weights in this group and therefore were not considered to be adverse. There were no test substance-related effects on mean body weights and body weight gains at 100 and 300 mg/kg/day or mean net body weights, net body weight gains, food consumption, or gravid uterine weights at 100, 300, and 500 mg/kg/day.

There were no test substance-related macroscopic findings noted at any dosage level.

Mean fetal body weights in the 1000 mg/kg/day group were up to 16.2% lower than the control group and corresponded to the lower mean gravid uterine weight observed in this group. Fetal survival in this group was not affected by test substance administration. Intrauterine growth and survival in the 100, 300, and 500 mg/kg/day groups were unaffected by maternal test substance administration. Test substance-related higher mean total proportion of developmental variations were noted in the 1000 mg/kg/day group when compared to the control group due to a test substance-related higher percent per litter of skeletal developmental variations observed in this group. Higher mean litter proportions of unossification of sternebra(e) no. 5 and/or 6 and sternebra(e) nos. 1, 2, 3, and/or 4 and reduced ossification of the vertebral arches were noted in the 1000 mg/kg/day group compared to the control group. The aforementioned skeletal developmental variations were considered test substance-related and secondary to the reduced fetal body weights noted at this dosage level. No test substance-related fetal malformations were observed at any dosage level and no test substance-related fetal developmental variations were noted at 100, 300, and 500 mg/kg/day.

Based on adverse clinical findings and mean body weight losses and lower mean body weight gains with corresponding decreased food consumption observed at 1000 mg/kg/day, a dosage level of 500 mg/kg/day was considered to be the no-observed-adverse-effect level (NOAEL) for maternal toxicity. Based on decreased mean fetal body weights with an associated increase in the mean litter proportions of skeletal developmental variations related to reduced ossification at 1000 mg/kg/day, a dosage level of 500 mg/kg/day was considered to be the NOAEL for embryo/fetal development when the test material was administered orally by gavage to bred Crl:CD(SD) rats. Test substance-related increased mean litter proportions of skeletal developmental variations consisted of reduced ossification or unossified bones which is indicative of developmental delay and was considered secondary to the reduced fetal body weights noted at this dosage level. The embryo/fetal effects were associated with maternal toxicity and the corresponding decrease in food consumption in the dams.

Reproduction / Developmental Toxicity Screening Test (OECD 421)

The study was conducted to generate limited information concerning the effects of the test item (reaction product of amines, C12-14,-tert-alkyl, alcohol, C14-18, C18 unsat, and phosphorus pentoxide) on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus, and parturition (Thorsrud, 2013). According to OECD Guideline 421, three treatment groups of twelve CD [Crl:CD(SD)] rats/sex/group were administered to the test article at dose levels of 150, 500, or 1000 mg/kg/day. One additional group of twelve animals/sex served as the control and received the vehicle, peanut oil (arachis oil NF). The vehicle or test article was administered to all groups daily via oral gavage at a dose volume of 2 mL/kg/dose. Dosing began 14 days prior to pairing and continued to euthanasia (43 days total) for the parental (P) males. 250 observations of the P animals included clinical signs, body weights and body weight change, and food consumption during the premating/mating, gestation, and lactation periods, and parturition and litter data. Observations of the offspring (F1) included survival at birth and during lactation, individual clinical signs, pup body weights and sex, and gross abnormalities. At study termination, necropsy examinations were performed on all surviving P animals, and organs and tissues were collected, weighed, and examined for select groups. Dams losing their entire litter were subjected to a necropsy, and up to ten pups from the litters were saved in fixative for possible future examination. On LD 4, surviving F1 pups were examined externally, euthanized, and discarded.

The administration of the test substance produced clinical observations in parental animals consisting of post dose salivation and yellow discoloured hair in both male and female animals predominantly at 500 and 1000 mg/kg/day. Additional clinical findings were noted in the females predominantly at 500 and 1000 mg/kg/day that included increased activity, few/absent faeces, and vulvar discharge. Two females at 1000 mg/kg/day had to be euthanized moribund close to the time of parturition. Test article-related changes in body weight and food consumption were limited to females at 500 and 1000 mg/kg/day. Test article-related macroscopic findings were present in the adrenal gland, thymus, and stomach of females at 1000 mg/kg/day. Test article-related organ weight changes were present in the ovaries, liver, kidneys, adrenal glands, and thymus at 500 and 1000 mg/kg/day. Test article-related microscopic findings were present in the ovaries, liver, kidneys, adrenal glands, stomach, and duodenum. Most findings were predominantly observed at 1000 mg/kg/day, but were also noted to some extent in animals at 500 mg/kg/day.

The F1 pup survival index and mean body weight were adversely affected by treatment with the test item at 500 and 1000 mg/kg/day. F1pup survival was statistically decreased at 500 and 1000 mg/kg/day as evidenced by significantly reduced viability indices (number of pups surviving to LD 4 divided by the number of live pups at delivery times 100) of 66.17 and 0.00%, respectively. In addition, at 1000 mg/kg/day there was a statistically significant decrease in the total number of pups born per litter on LD 0 (9.9 pups vs. 12. 9 pups in the controls) and a significant decrease in the number of live born pups per litter on LD 0 (6.6 vs. 12.9 in the controls). By LD 4, the number of live born pups per litter was statistically reduced at 500 mg/kg/day (9.0 pups vs. 12.8 pups in the controls). The decreases F1survival appears to be associated with considerable maternal toxicity, particularly at 1000 mg/kg/day. During the last week of gestation (GD 14-20), the dams at 1000 mg/kg/day were noted with mean body weight that was over 50% lower than controls and food consumption that was almost 20% lower than controls. Most of the F1offspring at 1000 mg/kg/day were either stillborn or died shortly after birth, with none of them surviving to LD 4. At 500 mg/kg/day the dams did not show any significant changes in body weight or food consumption during gestation (prior to delivery), but during the lactation period (LD 0 to 4) these dams did exhibit a significant decrease in mean food consumption, which was 27% lower than controls and a net body weight loss during this period. The decreased survival of the F1offspring at 500 mg/kg/day, in association with the maternal toxicity observed, may have been the result of either one or a combination of the following scenarios: decreased maternal care, an inability to produce enough milk, or offspring lacking the ability or interest to properly nurse. These changes in F1pup survival at 500 and 1000 mg/kg/day were considered to be test article related.

Based on the results obtained from this study, the No-Observed-Adverse-Effect-Level (NOAEL) for general toxicity in parental male and female animals was considered to be 150 mg/kg/day, which was generally based on, but not limited to, significant clinical findings, decreases in body weight and food consumption (primarily females), as well are organ weight changes and microscopic findings at 500 and 1000 mg/kg/day. A No-Observed-Effect-Level (NOEL) for F1offspring for developmental toxicity was considered to be 150 mg/kg/day, based on the significant decreases in survival and mean body weight at 500 and 1000 mg/kg/day. However, mating, fertility, and fecundity indices were unaffected by treatment with the test item at dose levels up to 1000 mg/kg/day, the highest dose level tested.


Justification for selection of Effect on developmental toxicity: via oral route:
Only one developmental study available.

Justification for selection of Effect on developmental toxicity: via inhalation route:
No relevant route of exposure.

Justification for selection of Effect on developmental toxicity: via dermal route:
The substance is neither a skin irritant nor a skin sensitiser. Thus, the evaluation of this endpoint is not justified.

Justification for classification or non-classification

Toxicity to reproduction

According to the classification criteria outlined in section 3.7.2.2 (Guidance on the Application of CLP criteria, 2012), if there is evidence from humans or experimental animals, possibly supplemented with other information, of an adverse effect on sexual function and fertility, or on development, the classification as Reproductive Toxicant is assigned. Mating, fertility, and fecundity indices were unaffected by treatment with the test item in the reproductive/development toxicity screening study (OECD 421). In the screening study, the NOAEL for reproductive performance was established to be 1000 mg/kg bw, the highest dose level tested. Reproductive function was also not affected in the developmental study (OECD 414): pre- and postimplantation losses, live litter size, and fetal sex ratios in the 1000 mg/kg/day group were unaffected by test substance administration; mean numbers of corpora lutea and implantation sites and the mean litter proportions of pre-implantation loss were similar across all treatment groups. Based on adverse clinical findings and mean body weight losses and lower mean body weight gains with corresponding decreased food consumption observed at 1000 mg/kg bw, a dosage level of 500 mg/kg bw was considered to be NOAEL for maternal toxicity.

Based on these results, this substance does meet the requirement under EU CLP (Regulation (EC) No. 1272/2008) for classification and labelling as a reproductive toxicant.

Developmental Toxicity

An assessement of developmental toxicity potential of the test substance (Reaction products of alcohols, C14-18, C18 unsat., esterified with phosphorus pentoxide and salted with amines, C12-14,-tert-alkyl) is based on the results of two studies: reproduction/developmental toxicity screening study (OECD 421) and developmental toxicity study (OECD 414).

Significant decreases in survival and mean body weight of pups in the mid and the highest (500 and 1000 mg/kg/day) dose groups were noted in the OECD 421 study. All pups died on day 0 in the highest dose group and some pups died prematurely in the mid dose group. The decreases in survival are attributed to considerable maternal toxicity and stress observed in female animals in these dose groups. It is evident from the following observations:

-      there was no effect on food consumption or weight gain in the males at 150, 500 or 1000 mg/kg/day while in females, there were significant reductions in food consumption and bodyweight gain in the 1000 mg/kg/day group and food consumption was decreased during lactation in the 500 mg/kg/day group. In this group, many pups did not survive up to lactation day 4. Therefore, the reduction in food consumption could have been secondary to the reduced number of pups that required feeding. In the 1000 mg/kg/day dose group all pups died on the day of birth;

-      there was a marked sex difference in terms of the effects of the test item on the adrenal glands and thymus. In males there were modest increases in relative adrenal gland weights at 500 and 1000 mg/kg/day but much greater increases in females. For the thymus weight there was only a minimal reduction at 1000 mg/kg/day in males but large dose-related decreases in the females at 500 and 1000 mg/kg/day. However, in the OECD 407 study the changes seen in these organs were much less marked. These organ changes are typically highly correlated with stress and it is not clear why there was such a difference between these two studies other than the fact that the females were pregnant in the OECD 421 study and that different strains of rat were used (CD rats in the 421 and Han Wistar in the 407);

-      there are no data in the OECD 421 report describing whether milk was observed in the stomachs of the pups on day 0 or day 4, although it is logical to assume that the reason that some pups died prematurely in the 500 mg/kg/day group and all pups died on day 0 in the 1000 mg/kg/day group, could have been related to poor maternal behaviour as a result of the stress following exposure to the test item. There was mention in the report about animals being cold to the touch. Stress is known to be a risk factor for adverse outcomes in pregnancy in mammals. On the balance of probabilities it may be assumed that the effects on the offspring are secondary to the stress effects on the females.

To resolve the question as to whether the effects observed are direct or indirect developmental toxicity, an OECD 414 study was conducted (Herberth, 2015). The selection of the dose levels for the developmental study was based on the results of the OECD 421 study. Treatment-related effects were observed at dosages of 500 and 1000 mg/kg/day following a duration of exposure that was more than twice as long as the duration in the developmental study. Body weight effects were limited to females. Occurrences of mean body weight decreases were sporadic during the early weeks of the study and did not become consistent and pronounced until the end of the gestation period. No animals were found dead during the course of the study, although 2 females in the 1000 mg/kg/day group were euthanized in extremis at the time of parturition on gestation days 21 or 22. The NOAEL was 150 mg/kg/day. Therefore, a high-dosage level of 1000 mg/kg/day and lower dosage levels were chosen to evaluate the dose-response of the test substance in the developmental study.

In this study, developmental toxicity was manifested at 1000 mg/kg/day. Mean male, female, and combined fetal body weights in the 1000 mg/kg/day group were statistically significantly lower (up to 16.2%) than the concurrent control group. Test substance-related increased mean litter proportions of skeletal developmental variations consisted of reduced ossification or unossified bones which is indicative of developmental delay and was considered secondary to the reduced fetal body weights noted at this dosage level. The developmental toxicity noted at the 1000 mg/kg/day dosage level occurred in the presence of maternal toxicity. Test substance-related statistically significantly mean body weight losses and lower mean body weight gains with corresponding lower mean food consumption were noted throughout the study. As a result, mean maternal body weights in the 1000 mg/kg/day group were 4.6 % to 19.4 % lower than the control group during gestation days 8-20. Mean gravid uterine weight, net body weight, and net body weight gain in this group were also statistically significantly lower than the control group. In addition, adverse test substance-related increased incidences of red material around the nose and mouth, decreased defecation, pale feces, and yellow material around the urogenital area were noted for the 1000 mg/kg/day group at the daily examinations and/or approximately 1 hour following dose administration generally throughout treatment period.

The ECHA CLP guidance indicates that it is difficult to disassociate the effects on the mother from any direct effects on the pups. However, the guidance also states: “Classification is not necessarily the outcome in the case of minor developmental changes, when there is only a small reduction in foetal/pup body weight or retardation of ossification when seen in association with maternal toxicity” and “Adverse effects on postnatal survival and growth seen only at dose levels causing maternal toxicity may be due to lack of maternal care or other causes such as adverse effects on or via lactation or developmental toxicity. In case "post-natal effects are caused by lack of maternal care classification for developmental effects may not be warranted,” also “an increased incidence of mortality among the treated dams over the controls shall be considered evidence of maternal toxicity if the increase occurs in a dose-related manner and can be attributed to the systemic toxicity of the test material. Maternal mortality greater than 10 % is considered excessive and the data for that dose level shall not normally be considered for further evaluation”. In the OECD 421 study 2 of 12 females were killed in extremis on GD 21 and 22 and a third female was found to have an incomplete delivery on GD 22. Therefore, the data from the 1000 mg/kg/day group should legitimately be excluded from any evaluation of developmental toxicity. In this case, it is proposed that no classification for developmental toxicity be applied. The developmental changes seen in this study at 500 mg/kg/day were primarily small reductions in the weight of the pups and were secondary to maternal toxicity that resulted in a lack of maternal care. In the OECD 414 study, decreased mean fetal body weights with an associated increase in the mean litter proportions of skeletal developmental variations related to reduced ossification were observed at 1000 mg/kg/day. Therefore, a dosage level of 500 mg/kg/day was considered to be the NOAEL for embryo/fetal development. However, test substance-related increased mean litter proportions of skeletal developmental variations which consisted of reduced ossification or unossified bones and which is indicative of developmental delay was considered secondary to the reduced fetal body weights noted at this dosage level. The embryo/fetal effects were associated with maternal toxicity and the corresponding decrease in food consumption in the dams.

Based on this knowledge, no direct developmental toxicity can be attributed to the test substance. The substance does not meet the requirement under EU CLP (Regulation (EC) No. 1272/2008) for classification and labelling as a developmental toxicant.

Additional information