2,6-dibromo-4-cyanophenyl octanoate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Source, RA-A, CAS 1689-84-5, M-227073-01-1; 2-generation reproductive toxicity (rat, similar to OECD 416, GLP):

NOAEL (P0 general parental toxicity): 50 ppm (corresponding to 3.39 mg/kg bw/day for males and 4.11 mg/kg bw/day for females, respectively)

NOAEL (P1 general parental toxicity): 50 ppm (corresponding to 3.98 mg/kg bw/day for males and 4.54 mg/kg bw/day for females, respectively)

NOAEL (F1 pup developmental toxicity): 50 ppm (corresponding to 3.39 mg/kg bw/day for males and 4.11 mg/kg bw/day for females, respectively)

NOAEL (F2 pup developmental toxicity): 50 ppm (corresponding to 3.98 mg/kg bw/day for males and 4.54 mg/kg bw/day for females, respectively)

NOAEL (P0 reproductive toxicity): 250 ppm (corresponding to 17.75 mg/kg bw/day for males and 21.5 mg/kg bw/day for females, respectively)

NOAEL (P1 reproductive toxicity): 250 ppm (corresponding to 21.3 mg/kg bw/day for males and 25.1 mg/kg bw/day for females, respectively)

 

LOAEL (P0 general parental toxicity): 250 ppm (corresponding to 17.75 mg/kg bw/day for males and 21.5 mg/kg bw/day for females, respectively)

LOAEL (P1 general parental toxicity): 250 ppm (corresponding to 21.3 mg/kg bw/day for males and 25.1 mg/kg bw/day for females, respectively)

LOAEL (F1 pup developmental toxicity): 250 ppm (corresponding to 17.75 mg/kg bw/day for males and 21.5 mg/kg bw/day for females, respectively)

LOAEL (F2 pup developmental toxicity): 250 ppm (corresponding to 21.3 mg/kg bw/day for males and 25.1 mg/kg bw/day for females, respectively)

 

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

refer to the analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at this dose level.

Remarks on result:

other: corresponding to approximately 3.39 mg/kg bw/day (males) and 4.11 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to approximately 17.75 mg/kg bw/day (males) and 21.5 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level (highest dose tested).

Remarks on result:

other: corresponding to approximately 17.75 mg/kg bw/day (males) and 21.5 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: corresponding to approximately 3.98 mg/kg bw/day (males) and 4.54 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Dose descriptor:

LOAEL

Remarks:

general toxicity

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

Remarks on result:

other: corresponding to approximately 21.3 mg/kg bw/day (males) and 25.1 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive toxicity

Effect level:

>= 250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects were observed at this dose level.

Remarks on result:

other: corresponding to approximately 21.3 mg/kg bw/day (males) and 25.1 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects observed at this dose level.

Remarks on result:

other: corresponding to approximately 3.39 mg/kg bw/day (males) and 4.11 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: physical development particularly with respect to eye opening

Remarks on result:

other: corresponding to approximately 17.75 mg/kg bw/day (males) and 21.5 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F2

Effect level:

50 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: corresponding to approximately 3.98 mg/kg bw/day (males) and 4.54 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Generation:

F2

Effect level:

250 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: physical development particularly with respect to eye opening

Remarks on result:

other: corresponding to approximately 21.3 mg/kg bw/day (males) and 25.1 mg/kg bw/day (females)

Remarks:

Source: CAS 1689-84-5, 1989, OECD 416, 2-Generation

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Study duration:

chronic

Species:

rat

Quality of whole database:

Toxicity to reproduction was assessed in a 2-generation study conducted with male and female rats similar to OECD TG 416 (1989), under GLP conditions (RL2). The study is considered of reliable quality and validity, fulfills the criteria of a key study and thus, is suitable for assessment of the present endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Data on male reproduction following percutaneous application is available for the target substance (CAS 1689-99-2) in rats. However, as dermal absorption is considered limited compared to oral absorption, the dermal route is not considered as the most appropriate route for hazard and risk assessment. Therefore, a read-across approach to the analogue substance (CAS 1689-84-5) was performed. The read-across is based on (bio) transformation to common compound(s), which thus have the same biological target(s) and therefore cause the same effects. A detailed justification for read-across is provided in the technical dossier (see IUCLID section 13.2). To assess the effect on reproduction of the test item, a 2-generation key study conducted with rats is available.

 

Oral route

In the 2-generation reproductive toxicity study (M-227073-01-1, 1989), male and female Sprague Dawley rats were fed with the source substance (CAS 1689-84-5) over two generations; all parental animals (P0 and P1) received the test substance via the diet throughout the study period. The study was performed under GLP conditions and similar to with OECD guideline 416 (1983). As the study is slightly dated, there are some noteworthy deviations to the current version of the OECD 416 (2001). Sperm parameters (except testes and epididymis weight) were not measured, the time point of vaginal opening and preputial separation was not determined for F1 pups. In addition, the anogenital distance was not measured. Histopathological examinations were performed on liver and kidney only.

24 male and 24 female Sprague-Dawley rats per group were continuously dosed with the test item via the diet throughout the entire study period at 0, 10, 50 or 250 ppm (corresponding to 0.69, 3.39 and 17.75 mg/kg bw/day (males) and 0.81, 4.11 and 21.5 mg/kg bw/day (females), respectively). Dosing started at ca. 6 weeks of age (first day of premating, P0 generation) and 3 to 4 weeks of age (post weaning, F1 generation) and continued through mating, gestation and lactation phases. Dose levels of the P1 generation were determined to be 0.77, 3.98, and 21.3 mg/kg bw/day (males) and 0.9, 4.54 and 25.1 mg/kg bw/day (females). Animals were observed for clinical signs and monitored for changes in body weight and food consumption. In addition, all adult animals were evaluated for treatment-related effects on estrous cycling, mating, fertility and gestation length. Litters were evaluated for effects on pup body weight, litter size, sex ratio, pup viability and physical development. P1 animals were evaluated as described for P0. Further examinations included a gross necropsy for all males, all dams following weaning and pups not selected to become F1 generation parents.

No remarkable clinical signs were observed during any phase of either generation. Statistically significantly reduced bodyweight gain was observed for P1 males, P0 and P1 females prior to pairing and during the first two-thirds of each gestation period, and F1A, F1B and F2 offspring during the lactation periods. Physical development of offspring reflected the slight retardation in bodyweight gain, particularly with respect to eye opening. Slight increases in relative liver weights were recorded for P0 males and P0 and P1 females, and in relative kidney weights for P1 males and females. There were, however, no adverse effects upon reproductive performance and fertility of either the P0 or P1 generation. Histopathological examination of tissues revealed no treatment-related changes.

In conclusion, the available study is reliable and valid and can be used as a key study. Based on the findings of this study, a NOAEL for general parental toxicity of 50 ppm for the P0 and P1 parental animals (corresponding to 3.39 and 4.11 mg/kg bw/day for P0 males and females, and to 3.98 and 4.54 mg/kg bw/day for P1 males and females, respectively) based on decreased body weights and increased liver weights. The NOAEL for effects on neonatal parameters was also 50 ppm (corresponding to 3.39 and 4.11 mg/kg bw/day for F1 males and females and 3.98 and 4.54 mg/kg bw/day for F2 males and females, respectively) based on decreased pup body weight and delayed physical development, especially regarding eye opening. The NOAEL for reproductive effects was ≥250 ppm (corresponding to 17.75 and 21.5 mg/kg bw/day for P0 males and females and 21.3 and 25.1 mg/kg bw/day for P1 males and females, respectively), the highest dose tested.

An additional report calculated the benchmark doses (BMD) and their confidence limits (lower 5% limit BMDL, upper 95% limit BMDU) based on the described 2-generation study in rats (M-499700 -01-1). The endpoints used in this evaluation were body weight of females (pre-mating Week 3, 6, 9 and 14; Day 20 of gestation; and Day 21 of lactation), body weight of pups (Day 21) and time point of eye-opening of pups. Benchmark dose calculations followed EFSA (2009a). The results can be found in Table 1 and 2 in Attachment 1 of the attached background material.

 

Dermal route

In addition to the oral route, reproductive toxicity following dermal application of the target substance was investigated. The dermal route is not considered as the most appropriate route for hazard and risk assessment according to the standard data requirements of Regulation EC (No.) 1907/2006, as dermal penetration of the target substance is approx. 50% (please refer to the data provided under “Toxicokinetics”) compared to the oral route. Further, the dermal acute or repeated dermal dose toxicity studies clearly suggest that systemic availability of the test substance was limited compared to that via the oral route. Therefore, the available data on dermal exposure to the target substance is considered as supporting evidence for the outcome of the oral studies performed with the source substance, thereby confirming the analogue approach (for further information, please refer to the detailed justification for read-across provided in the technical dossier (see IUCLID section 13.2)).

In the supporting study, Sprague-Dawley rats were dermally treated with the target substance (CAS 1689-99-2) for three weeks and the effects on male reproduction were examined (M-227334-01-1, 1990). The study was conducted according to EPA OPP 83-4 and in compliance with GLP standards.

Groups of 125 male rats/dose received the test substance dermally for 21 consecutive days at doses of 25, 50 and 100 mg/kg bw/day.Two additional groups were treated either with water (vehicle control group) or a Blank solution (blank control group). The solutions were prepared at concentrations of 0 (vehicle and blank), 25, 50, 100 mg/mL.The test substance was provided in the form of the Buctril formulation (33.8% test substance). The blank was a formulation of solvents and surfactants diluted to concentrations equivalent to those in the 25 mg/mL dose level of the test substance. The formulations were applied to a 20 cm² shaved area on the back at a dosage volume of 1 mL/kg bw for 6 h. During the dosing period, animals were observed for skin reactions, changes in body weight, food consumption and signs of ill-health.

Prior to dosing, 20 male rats per dose group were assigned randomly to participate in seven mating trials on postdosage days 1, 7, 14, 21, 35, 56 and 113. Each of these males was assigned one untreated virgin female per mating trial (5 days with the same virgin female rat). On Day 14 of presumed gestation the female rats were sacrificed and examined for pregnancy status and the number and placement of implantations, resorptions and live and dead embryos. The 20 males per group were sacrificed after the last mating trial (Day 119 postdosage). The remaining 105 male rats per dose group were randomly selected for interim sacrifices including testicular evaluations on Days 1, 7, 14, 21, 35, 56 and 113 postdosage.

No treatment-related death occurred during the course of the study. One rat of the Blank dose group that was scheduled for sacrifice on Day 35 postdosage was found dead on Day 15 postdosage. This finding was not considered treatment related. No clinical signs of toxicological relevance were observed in any treatment or control group, neither in male nor in female rats. The vehicle control group showed no skin reactions throughout the dosing period whereas treatment with the blank and the test substance formulations caused erythema and desquamation. The onset of effects was generally after one or two dosages had been applied, and the severity of the skin reactions was dosage-dependent, increasing with continued application. In the 25 mg/kg bw/day dose group, skin reactions were observed during the dosing and postdosage period, but the number of animals showing these reactions reached statistical significance only during the postdosage observations. For all other treatment groups as well as the blank group, the number of animals with skin reactions to treatment were statistically significantly increased during the administration and the postdosage period. In general, the numbers of rats with some degree of skin irritation caused by each dosage of the test substance were greater than those in the group given the blank, although similar number of rats in the 0 (Blank) and 100 mg/kg bw/day dosage groups had the most severe reaction observed, grade 4 erythema (severe redness, scabs and/or cracks). Evidence of the skin reactions caused by the blank and 25, 50 and 100 mg/kg bw/day dosages of the test substance gradually disappeared during the postdosage period.

The average body weight gains were statistically significantly reduced for the groups receiving 50 and 100 mg/kg bw/day of the test substance during the dosage period compared to the vehicle control and the terminal body weight differed statistically significantly from that of controls for the high dose group. No effects on body weight or body weight gain were observed in the 0 (Blank) and 25 mg/kg bw/day groups. The 100 mg/kg bw/day dose group had a significant increase in the absolute feed consumption value on days 8 to 15 of dosage for the group sacrificed on Day 7 postdosage. No other statistically significant differences occurred in absolute feed consumption values during the dosage or postdosage periods. Relative feed consumption was increased statistically significantly as compared to animals of the vehicle control groups for the 50 and 100 mg/kg bw/day dose groups during the dosage period and for the 25, 50 and 100 mg/kg bw/day dosage groups during the first and/or second weeks of the postdosage period.

No gross lesions in the male rats were caused by dermal administration of the blank or the test substance up to the highest dose tested.

Considering reproductive parameters, no relevant differences were found between control and treatment groups. The averages for days in cohabitation, the numbers of rats mating and not mating, the numbers of rats with or without confirmed mating dates, and the numbers of matings that resulted or did not result in pregnancy were similar across all groups. Similarly, there were no differences between the groups with regard to the averages of corpora-lutea, implantations, live and dead fetuses and early and late resorptions revealed at Caesarean-sectioning of the mated female rats. Normal sperm per rat, percent of abnormal sperm, the various types of abnormalities in sperm morphology, averages for total motile sperm, total nonmotile sperm or total percent motile sperm or total epididymal sperm concentrations were not affected by treatment with the test substance or the Blank. The observation of nonmotile sperm that was observed in some rats, occurred without dosage-dependency and were considered unrelated to the test substance. In terms of testis weight or testicular spermatid concentration, no differences between control and treatment groups were found.

The averages for liver weight relative to body weight were significantly increased for the high dose group at sacrifice on Days 1 and 7 postdosage. Since this was not seen in groups sacrificed later, it was considered an adaptive effect.

No other differences in the averages for terminal body weights or absolute or relative weights of the left epididymis, left cauda epididymis, left or right testis, prostate, seminal vesicles with and without fluid, pituitary and liver were considered effects of the test substance.

Histopathological examination of the seminiferous tubules of the right testis of the vehicle, blank and high dose group male rats for the 19 stages of spermatogenesis did not reveal any effects of the test substance. Microscopic examination of the caput, corpus and cauda regions of the epididymis of all male rats did not reveal dosage-dependent or statistically significant alterations.

Based on the findings of this study, a LOAEL of 25 mg/kg bw/day for P males was set for dermal irritation (local effects) based on skin irritation during the dosage and postdosage periods. A NOAEL of 25 mg/kg bw/day for general toxicity was set based on altered body weight and increased relative feed consumption values during the postdosage period observed at 50 mg/kg bw/day.The reproductive NOAEL for the test substance was set at ≥100 mg/kg bw/day, the highest dose tested. Significant reductions in the absolute and relative prostate weights occurred in the rats of the 100 mg/kg bw/day group sacrificed on Day 1 postdosage, observations interrelated with the reduced body weight of this group. No effects on reproductive performance, male reproductive organ weights other than the prostate, testicular endpoints or histopathology were observed after application of the test substance up to the highest dose level.

 

Conclusion on reproductive toxicity
In the present study, the effect of the source substance on reproduction in Sprague Dawley rats through two successive generations was investigated. The study showed that the source substance had no adverse effects on mating performance, sexual function, fertility, lactation or the outcomes of the pregnancies.Slight delays in neonatal growth were seen at the higher doses but they did not affect the animals' maturation to adulthood and no physical evidence for such an effect was present at further physical examination or at necropsy. The evidence suggests that there were no demonstrable physical anomalies present that affected the offspring's capability to successfully produce a second generation. The data clearly suggested that, at the treatment levels studied, the test substance did not adversely affect the development of the offspring treated continuously in utero, via lactation and later via their diet. Hence, no classification or labelling according to the CLP Regulation 1272/2008, would be required with regard to reproductive toxicity, i.e. toxicity to sexual function and fertility.

This was also supported by the study on male fertility and reproductive function after dermal application of the target substance.In this study, the reproductive NOAEL for the test substance was set at ≥100 mg/kg bw/day, the highest dose tested. Significant reductions in the absolute and relative prostate weights occurred in the rats of the 100 mg/kg bw/day group sacrificed on Day 1 postdosage. These observations interrelated with the reduced body weight of this group. No effects on reproductive performance, male reproductive organ weights other than the prostate, testicular endpoints or histopathology were observed after application of the test substance up to the highest dose level. Hence, no classification or labelling according to the CLP Regulation 1272/2008, would be required with regard to male reproductive toxicity.

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.

Effects on developmental toxicity

Description of key information

Source, RA-A, CAS 1689 -84 -5, Developmental toxicity (rat, similar to OECD 414, GLP):

NOAEL (maternal systemic toxicity): 12.5 mg/kg bw/day

LOAEL (maternal systemic toxicity): 40 mg/kg bw/day

NOAEL (developmental toxicity): 4 mg/kg bw/day

LOAEL (developmental toxicity): 12.5 mg/kg bw/day

 

Source, RA-A, CAS 1689 -84 -5, Developmental toxicity (rabbit, no guideline, non-GLP):

NOAEL (maternal systemic toxicity): 15 mg/kg bw/day

LOAEL (maternal systemic toxicity): 30 mg/kg bw/day

NOAEL (developmental toxicity): 30 mg/kg bw/day

LOAEL (developmental toxicity): 60 mg/kg bw/day

 

Source, RA-A, CAS 1689-84-5, 2-generation reproductive toxicity (rat, similar to OECD 416, GLP):

NOAEL (P0 general parental toxicity): 50 ppm (corresponding to 3.39 mg/kg bw/day for males and 4.11 mg/kg bw/day for females, respectively)

NOAEL (P1 general parental toxicity): 50 ppm (corresponding to 3.98 mg/kg bw/day for males and 4.54 mg/kg bw/day for females, respectively)

NOAEL (F1 pup developmental toxicity): 50 ppm (corresponding to 3.39 mg/kg bw/day for males and 4.11 mg/kg bw/day for females, respectively)

NOAEL (F2 pup developmental toxicity): 50 ppm (corresponding to 3.98 mg/kg bw/day for males and 4.54 mg/kg bw/day for females, respectively)

NOAEL (P0 reproductive toxicity): 250 ppm (corresponding to 17.75 mg/kg bw/day for males and 21.5 mg/kg bw/day for females, respectively)

NOAEL (P1 reproductive toxicity): 250 ppm (corresponding to 21.3 mg/kg bw/day for males and 25.1 mg/kg bw/day for females, respectively)

 

LOAEL (P0 general parental toxicity): 250 ppm (corresponding to 17.75 mg/kg bw/day for males and 21.5 mg/kg bw/day for females, respectively)

LOAEL (P1 general parental toxicity): 250 ppm (corresponding to 21.3 mg/kg bw/day for males and 25.1 mg/kg bw/day for females, respectively)

LOAEL (F1 pup developmental toxicity): 250 ppm (corresponding to 17.75 mg/kg bw/day for males and 21.5 mg/kg bw/day for females, respectively)

LOAEL (F2 pup developmental toxicity): 250 ppm (corresponding to 21.3 mg/kg bw/day for males and 25.1 mg/kg bw/day for females, respectively)

 

Link to relevant study records

Reference

Reference 1

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:

refer to the analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Species:

rat

Key result

Dose descriptor:

NOAEL

Remarks:

maternal general toxicity

Effect level:

12.5 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level.

Remarks on result:

other: Source: CAS 1689-84-5, 1987, rat, OECD 414

Key result

Dose descriptor:

LOAEL

Remarks:

maternal general toxicity

Effect level:

40 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Source: CAS 1689-84-5, 1987, rat, OECD 414

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

4 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: no adverse effects observed at this dose level.

Remarks on result:

other: Source: CAS 1689-84-5, 1987, rat, OECD 414

Key result

Dose descriptor:

LOAEL

Remarks:

maternal developmental toxicity

Effect level:

12.5 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Remarks on result:

other: Source: CAS 1689-84-5, 1987, rat, OECD 414

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

Increased post-implantation loss was observed at 12.5 mg/kg bw/day. Maternal body weight nett of the gravid uterus was depressed among animals dosed at 40 mg/kg bw/day, indicating that the maternal compartment of the maternal-fetal complex was affected at this dosage.

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

4 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: Source: CAS 1689-84-5, 1987, rat, OECD 414

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

12.5 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Remarks on result:

other: Source: CAS 1689-84-5, 1987, rat, OECD 414

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: rib

Description (incidence and severity):

increases in the incidence of 14th thoracic ribs (further details are summarised in Attachment S)

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

12.5 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Reference 2

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:

refer to the analogue justification provided in IUCLID section 13.

Reason / purpose for cross-reference:

read-across source

Species:

rabbit

Key result

Dose descriptor:

NOAEL

Remarks:

maternal general toxicity

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects observed at this dose level.

Remarks on result:

other: Source: CAS 1689-84-5, 1983, rabbit

Key result

Dose descriptor:

LOAEL

Remarks:

maternal general toxicity

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Source: CAS 1689-84-5, 1983, rabbit

Key result

Dose descriptor:

NOAEL

Remarks:

maternal developmental toxicity

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects in regard to maternal developmental toxicity observed up to this dose level.

Remarks on result:

other: Source: CAS 1689-84-5, 1983, rabbit

Key result

Dose descriptor:

LOAEL

Remarks:

maternal developmental toxicity

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

pre and post implantation loss

Remarks on result:

other: Source: CAS 1689-84-5, 1983, rabbit

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

increased post-implantation loss at 60 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse findings at this dose level observed.

Remarks on result:

other: Source: CAS 1689-84-5, 1983, rabbit

Key result

Dose descriptor:

LOAEL

Remarks:

developmental toxicity

Effect level:

60 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

external malformations

skeletal malformations

visceral malformations

other: increased incidences of malformations (anophthalmia and microphthalmia, hydrocephalus and ossification effects) and supernumerary ribs

Remarks on result:

other: Source: CAS 1689-84-5, 1983, rabbit

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: eye

skeletal: supernumerary rib

visceral/soft tissue: urinary

visceral/soft tissue: eye

Description (incidence and severity):

Reduced fetal weight, increased incidences of malformations (anophthalmia and microphthalmia, hydrocephalus and ossification effects) and supernumerary ribs observed at 60 mg/kg bw/day.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

4 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Developmental toxicity was assessed in several prenatal toxicity studies conducted in rats and rabbits. The rat key study was performed according to OECD guideline 414 (1981) under GLP conditions. The study in rabbits was not conducted under GLP conditions and no guideline is mentioned but the overall quality of the study is considered as reliable. Further, toxicity to reproduction was assessed in a 2-generation study conducted in rats according to OECD TG 416 (1983) under GLP conditions. The studies are considered of reliable quality and validity, fulfill the criteria of key studies and thus, are suitable for assessment of the present endpoint.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

10 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

42

Species:

rat

Quality of whole database:

Two GLP compliant studies performed with the target substance according to U.S. EPA Pesticide Assessment Guideline; Subdivision F, Hazard Evaluation: Human and Domestic Animals, November 1984, 83-3, Teratology Study are available.

Additional information

Data on developmental toxicity following percutaneous application is available for the target substance (CAS 1689-99-2) in rats and rabbits. However, as dermal absorption is considered limited compared to oral absorption, the dermal route is not considered as most appropriate route for hazard and risk assessment. Therefore, a read-across approach to the source substance (CAS 1689-84-5) is applied in regard to developmental toxicity following oral administration. As discussed in the chapter on toxicokinetics, the target substance is converted to the source substance and hence, the read-across is based on (bio) transformation to common compound(s), which thus have the same biological target(s) and therefore cause the same type of effects. A detailed justification for read-across is provided in the technical dossier (see IUCLID section 13.2).

 

Data on developmental toxicity following oral and percutaneous administration are available for the source substance to characterize the potential to induce toxicity after in utero exposure in rodents and non-rodents. Two key studies are available addressing developmental toxicity of the test substance in the rat and rabbit after oral administration. Further, developmental toxicity was evaluated in the rat and rabbit following dermal administration. Supporting evidence on developmental toxicity is available based on a mechanistic study in rats.

 

Developmental toxicity studies via the oral route

Rat

One preliminary teratology study was performed in the rat as dose range-finding study (M-344550-01-2, 1985). 6 mated female CD rats were exposed to 0 (0.5% carboxymethyl cellulose), 5, 15 or 45 mg/kg bw/day with the source substance (CAS 1689-84-5). Four additional rats were dosed at 140 mg/kg bw/day. The test suspension was prepared daily in 0.5% carboxymethyl cellulose and administrated by oral gavage, from day 6 to day 15 of gestation (day plugs and vaginal sperm found = day 0). The dose administered daily to each animal was based on the animal’s bodyweight on that day. All animals were observed from day 0 (start of experiment) to day 20 of gestation. Health and clinical signs were recorded daily, maternal body weight was recorded on days 0, 3, 6 - 15, 17 and 20 of gestation, maternal food consumption was recorded twice per week. On day 20 of gestation, females were sacrificed and examined for macroscopic abnormality of the reproductive tract, number of corpora lutea in each ovary, weight of gravid uterus, distribution of live and dead fetuses and distribution of resorption sites in each uterine horn, individual placental weights, individual fetal weights and sexes. Statistics based on Student's t-test using pooled within group error variance performed on data of maternal body weight, maternal food consumption, fetal weight and length, pre-implantation loss (transformed data), post implantation loss (transformed data). Fisher exact probability test was performed on data of maternal gross observations signs and mortality. Mann-Whitney U-test was performed on data of placental weight.

All females of the 140 mg/kg bw/day dose group died within 3 days of treatment. The animals had haemorrhagic lungs. All other animals in the other dose groups survived the entire study period and did not show any adverse clinical signs related to treatment. In animals of the lower dose groups, at 15 and 45 mg/kg bw/day reduced food consumption was observed. Body weight gain (total and nett of gravid uterus) was significantly depressed among animals in the 45 mg/kg be/day dose group. In regard to reproductive and fetal parameters, the following observations were made: Fetal and embryonic viability was not adversely affected by treatment, whereas fetal body weight and fetal length were depressed. Placentas at 45 mg/kg bw/day were larger than those in the control group and showed prominent crater-like depressions in their admetral surface. Based on the results of this study, the doses for the key study (M-344549-01-2, see below) were selected. The dose-range finding study was not conducted under GLP or any OECD guideline.

In the key developmental toxicity study (M-344549-01-2, 1987), which was conducted under GLP conditions and in accordance to OECD guideline 414 (1981), 22 female Sprague-Dawley rats per dose were co-housed with male rats (1/1 ratio) for mating. When pregnancy was assumed, pregnant dams were treated with the source substance (CAS 1689-84-5) at levels of 0 (0.5% carboxymethyl cellulose 0.5% acetic acid), 4, 12.5 and 40 mg/kg bw/day via gavage once daily from day 6 to day 15 of gestation (day plugs and vaginal sperm found = day 0). The dose administered daily to each animal was based on the animal’s body weight on that day.

All animals were observed from day 0 (start of experiment) to day 20 of gestation. General health and clinical signs were recorded daily. Maternal body weight was recorded on days 0, 3, 6 - 15, 17 and 20 of gestation. Maternal food consumption was recorded twice per week. On day 20 of gestation, females were sacrificed and examined for macroscopic abnormality of the reproductive tract, number of corpora lutea in each ovary, weight of gravid uterus, distribution of live and dead fetuses and distribution of resorption sites in each uterine horn, individual placental weights, individual fetal weights and sexes, external anomalies of individual fetuses, internal examinations of fetuses. One half of the fetuses per litter was examined for tissue alterations and the other for skeletal alterations. Statistical analysis relied on: Fisher exact test on maternal gross observational signs during gestation; Student's t-test using pooled within group error variance on maternal food consumption, body weight, uterine, fetal and placental weights, fetal length, number of corpora lutea and live fetuses and percent pre- and post-implantation loss; Chi-square test on fetal and skeletal examinations; Mann-Whitney U- test on litter mean proportions and Fisher exact test or Chi-square test on numbers of litter with one or more affected fetuses.

Analytical analyses revealed that the active ingredient was stable over 4 h in the dosage form used. All animals survived until scheduled sacrifice. Treatment-related effects were observed in females at 40.0 mg/kg bw/day, including markedly reduced food consumption (p<0.05) from day 7 to 20. Maternal body weight gain among high dosage animals was depressed in comparison to control (p<0.05, from day 0 to 20 and from day 6 to 20 corresponding to 12.6% and 14.4% respectively). Further, maternal body weight nett of the gravid uterus was also depressed among animals dosed at 40 mg/kg bw/day, indicating that the maternal compartment of the maternal-fetal complex was affected at this dosage.

Post-implantation loss was elevated in the 12.5 and 40.0 mg/kg bw/day dose groups, and in the latter group, fetal weight and fetal length were significantly reduced. In the group receiving 40 mg/kg bw/day, fetal abnormalities were more frequent, often indicative of generalized developmental retardation, consistent with reduced fetal body weight and length. These included shortened renal papillae and unossified vertebral centra, sternebrae, metacarpals and metatarsals. The occurrence of lumbar ribs among high and intermediate dosage fetuses, while not a major malformation, does indicate a deleterious effect of the test compound upon morphogenesis. Fetuses of the 40 mg/kg bw/day group showed higher incidence of anophthalmia and microphthalmia. If data from fetuses allocated to free-hand sectioning and those allocated to skeletal evaluation are pooled, the overall incidence of anophthalmia/microphthalmia in the high dosage group is 19/319, or 6%. In fetuses of the high dose group, additional malformations were observed. These included diaphragmatic hernia, narrowing of the aortic arch and fusion of ribs associated with scoliosis (the last two, in one case associated with esophageal atresia and absence of the trachea). While none of these observations taken alone achieved statistical significance in comparison to controls, their overall pattern of incidence is suggestive of treatment relation. One fetus of the 12.5 mg/kg bw/day group also showed pronounced narrowing of the aortic arch, a treatment-dependency cannot be excluded.

In one fetus of the 4 mg/kg bw/day group, anophthalmia was also observed. However, this fetus also showed hydrocephalus and a right aortic arch. In contradistinction to anophthalmic and microphthalmic fetuses in the high dosage groups, this low dosage fetus was dramatically runted. This fetus was found at the distal (ovarian) end of the uterus, an implantation site often associated with reduced fetal size. Thus, it would be difficult to attribute the malformations observed in this fetus solely to the action of the test material. All placentae examined from control and high dosage groups, including one placenta described at necropsy with ‘irregular depressions on admetral surface’ were histologically normal.

In conclusion the available study is considered reliable and valid and is thus considered as a key study. The study was conducted according to OECD TG 414 with deviations regarding missing examinations recommended in the current version (2018). Based on the results it may be concluded that the test substance was found to be more toxic to fetuses than to pregnant female rats. Maternal animals were affected at the highest dose of 40 mg/kg bw/day, evident by reduced body weight, feed consumption and body weight nett of the gravid uterus. In the 12.5 and 40 mg/kg bw/day groups, visceral and skeletal malformations of fetuses were observed. Therefore, the NOAEL for maternal (systemic) toxicity was determined to be 12.5 mg/kg bw/day, whereas the NOAEL for fetal development was set to 4 mg/kg bw/day.

 

A further study on developmental toxicity on the source substance is available, which was not conducted under GLP conditions or according to any guideline (M-184740-01-1). However, it was performed similarly to the OECD guideline 414 (adopted 1981). 28 pregnant female Sprague-Dawley rats were treated with the source substance (CAS 1689-84-5) at levels of 0 (vehicle, 0.25% aqueous gum tragacanth.), 5, 15 and 35 mg/kg bw/day via gavage once daily from Day 5 – 17 post-coitum. All surviving animals were sacrificed on Day 22 post-coitum. Bodyweight and food consumption data for all rats were recorded to the nearest gram upon receipt and on days 5, 8, 11, 14, 18 and 22 post-coitum. All animals were observed daily, and any animals dying or found dead were subjected to a gross necropsy unless this was prevented by cannibalism or autolytic degeneration. The rats were killed on day 22 post-coitum, and subjected to a gross necropsy. The numbers of corpora-lutea, viable foetuses and early and late uterine deaths were recorded. Early uterine deaths were defined as those implantations without, and late deaths those with embryonal or foetal elements. Individual viable foetuses were weighed, sexed and examined in detail for external abnormalities and by dissection, after an intrathoracic injection of sodium pentobarbitone solution. Any abnormalities of placentation or amniotic structure were noted. The brains of all viable foetuses were examined by free-hand sectioning after fixation in Bouin 's fluid, and the skeletons by examination under a dissecting microscope after staining with alizarin red S. Statistical evaluation of the results was by means of: Students "t" test (food consumption, numbers of corpora lutea, number of implantations, numbers of viable foetuses and mean foetal body weight), Mann-Whitney "U" test (gestational bodyweight and bodyweight gain) and Chi squared test (pre-implantation loss, post-implantation loss, foetal sex ratio, numbers of foetal major malformations, minor anomalies and variants). A value of P of 0.05 or less was taken as the criterion of statistical significance. Dosing suspensions were analysed and were found to be accurate. Dosing preparations found to be stable following 10 weeks storage at -20°C.

6 animals of the high dose group were found dead prior to study termination. At least five of these deaths were considered to be compound-related. Statistically significant (p<0.01) decreases in mean bodyweight gain occurred during the dosing period (days 5-18 post-coitum) in the 15 and 35 mg/kg bw/day dose groups. In the 5 mg/kg bw/day group, slight decreases in body weight gain (11%, Days 5-14) occurred, but no statistically significance was derived. Statistically significant decreases in food consumption occurred at 35 mg/kg bw/day between Day 5 and 8 and 8 and 11. Pregnancy parameters were very similar between groups, no abortion, unusual pregnancy length or premature deliveries were reported. However, pregnancy data were not available from the six animals in the 35 mg/kg bw/day group that died. Although the mean numbers of implantations per animal were reduced at 35 mg/kg bw/day, inter group differences were statistically comparable. Mean pre-implantation losses were statistically significantly increased (P<0.05) at 35 mg/kg bw/day, but remained statistically comparable with the vehicle controls at 5 and 15 mg/kg bw/day. The mean numbers of viable foetuses per litter treatment groups were comparable statistically with the vehicle controls. However, there was a slight reduction in the mean number of viable foetuses at 35 mg/kg bw/day group litters. There were no statistically significant differences in the total number of uterine deaths at any dosage tested. The numbers of early uterine deaths associated with treatment were either comparable or less than those of the vehicle control group. However, late uterine deaths showed a statistically significant increase (P<0.05) at 35 mg/kg bw/day. Post-implantation losses (%) were statistically comparable with those of the vehicle control group. A statistically significant reduction (P<0.001) in foetal weight was observed in the 35 mg/kg bw/day litters. Treatment did not affect foetal weight at any other dosage tested. Neither the type nor the incidence of major malformation suggested any association with treatment at any dosage. However, in comparison with the vehicle controls there was a dose related increase in the incidence of minor anomalies associated with treatment, and, although not statistically significant this may possibly be of some biological significance. Skeletal variants, although routinely recorded are not normally reported unless of any significance. A dose-related increase in the incidence of the supernumerary 14th rib variant was associated with treatment. Statistical evaluation revealed that the number of foetuses exhibiting a 14th rib variant was significantly increased, (P<0.001) at both 35 and 15 mg/kg bw/day, when compared with the vehicle controls. Although the number of foetuses exhibiting a 14th rib variant was increased at 5 mg/kg bw/day, this was not significant statistically, but was far higher than the normal suggested by available background data for this strain of rat. In conclusion, in utero exposure to 35 mg/kg bw/day was directly associated with five maternal deaths, a marked reduction in bodyweight gain during the dosing and post-dosing periods and a reduction in food consumption during the initial part of the dosing period. Reduction in foetal weight, increased pre-implantation losses and increased late uterine deaths observed at 35mg/kg bw/day may have been related to a direct effect upon the embryo/foetus, although maternal toxicity could well have been a contributory factor. Reductions in observed maternal bodyweight gain at 15 mg/kg bw/day must also be concluded as being compound-related. The non-statistically significant but dose related trend in minor foetal anomalies and the statistically significant dose related trend in the supernumerary 14th rib variant may both be regarded as being compound related. Based on slight decreases, however not statistically significant, in body weight gain at 5 mg/kg bw/day the maternal NOAEL was < 5 mg/kg bw/day. Further, based on slight, however not statistically significant increase in the number of fetuses with extra ribs variants noted in the 5 mg/kg bw/day dose group, the developmental NOAEL was < 5 mg/kg bw/day.

 

Rabbits

Developmental toxicity following oral administration was further investigated in rabbits (M-184736-01-2, 1983). The study was not performed according to GLP and no testing guideline was followed. However, based on overall quality and similarities to OECD guideline 414, the study is considered to be valid and is used as a key study. A minimum of 15 pregnant female New Zealand White rabbits were treated with the source substance (CAS 1689-84-5) at levels of 15, 30 and 60 mg/kg bw/day via gavage once daily from days 5 – 20 post-coitum. An additional vehicle control received the concurrent vehicle (aqueous suspension containing 0.25% w/v gum tragacanth). Positive control animals received thalidomide (150 mg/kg bw/day) orally via gelatin capsules (10 animals). Bodyweights and food consumption were recorded on days 0, 5, 9, 13, 17, 21, 25 and 29 post-coitum. Between scheduled weighing days food hoppers were topped up with known amounts of food as necessary. All animals were observed at least once daily, and any animal dying or killed for humane reasons during the study was subjected to a gross necropsy. Rabbits killed on day 29 post-coitum were subjected to a macroscopic post-mortem examination and their uteri and ovaries removed. The numbers and positions of corpora lutea, viable foetuses and early and late uterine deaths were recorded. Early uterine deaths were defined as those implantations without, and late deaths as those with, embryonal or fetal elements. Viable fetuses were weighed and examined in detail for abnormalities, both externally and by dissection after sacrifice. Any abnormalities of placentation or amniotic structure were noted. Where possible the brains of all viable foetuses were examined by free-hand sectioning after fixation in Bouin' s fluid. The hearts of all viable foetuses were examined by dissection after fixation in formol saline. Skeletons were examined after staining with alizarin red S. Following fixation, foetal or maternal tissues showing any abnormality were selected for microscopic examination and routinely processed, sectioned and stained with haematoxylin and eosin.

No adverse clinical signs or mortalities were clearly associated with oral administration of the test substance. 2 animals of the high dose group were found dead an Day 13 and 23 post-coitum, respectively. At 15 mg/kg bw/day, a slight but not statistically significant reduction in mean body weight gain was observed, while body weight gain was significantly reduced in animals of the 30 and 60 mg/kg bw/day group compared to the vehicle control. The reduction lasted throughout the dosing period and was accompanied by decreased food consumption (throughout the dosing period for the high dose group and from Days 9 - 13 and 13 – 17 in the mid dose group). When treatment was terminated, animals of the mid and high dose group showed increased food consumption (post-dosing recovery), and the body weight increased, but it still differed from vehicle controls at study termination.

No differences in pre-implantation loss were observed among the groups. In the top dose group increased post-implantation loss and significantly lower fetal body weights were found (-18.1%). In the 30 mg/kg bw/day group, slight, non-significant increases in post-implantation loss occurred. The effect on post-implantation loss was primarily due to an increase in early uterine deaths. Fetuses of the 60 mg/kg bw/day group showed increases in major malformations (anophthalmia and microphthalmia, hydrocephalus and ossification effects). An increase in the incidence of supernumerary ribs occurred in the 60 mg/kg bw/day dose group. Slight, non-statistically significant increases in supernumerary ribs occurred in the 15 and 30 mg/kg bw/day dose groups. A higher frequency of fetuses with major malformations was observed in the thalidomide group compared to the vehicle control demonstrating the sensitivity of the strain to a known teratogen.

In conclusion, administration of the test substance to pregnant rabbits at 30 and 60 mg/kg bw/day was directly associated with reductions in gestational bodyweight gain and food consumption during the dosing period; there was little evidence of these reductions in the 15 mg/kg bw/day group. Thus, a maternal NOAEL for systemic toxicity of 15 mg/kg bw/day is derived. Further, the test substance was embryotoxic and teratogenic at 60 mg/kg bw/day and exhibited some degree of foetotoxicity as indicated by reduced fetal weight at termination of the study. There was no evidence of embryotoxicity, foetotoxicity or teratogenicity at dosages of 15 or 30 mg/kg bw/day and hence, a developmental NOAEL of 30 mg/kg bw/day is considered.

The study is considered reliable and valid.

 

Developmental toxicity studies via the dermal route

In addition to the oral route, developmental toxicity following dermal application of the target and source substance was investigated. The dermal route is not considered as most appropriate route for hazard and risk assessment according to the standard data requirements of Regulation EC (No.) 1907/2006, as dermal penetration of the target substance is in the region of approx. 50% (please refer to the data provided under “Toxicokinetics”) compared to the oral route. Further, the dermal acute or repeat dermal dose toxicity studies clearly suggest that systemic availability of the test substance was limited compared to that via the oral route. Therefore, the available data on dermal exposure to the target substance is considered as supporting evidence for the outcome of the oral studies performed with the source substance, thereby confirming the analogue approach (for further information, please refer to the detailed justification for read-across provided in the technical dossier (see IUCLID section 13.2)).

 

Rat

Developmental toxicity following percutaneous application of the target substance (CAS 1689-99-2) was investigated in Charles River Crl:CD(SD)BR rats (25 females/group) according to GLP and US EPA Pesticide Assessment Guidelines. Subdivision F, 83-3 (adopted 1984) (M-227387-01-1). The test substance was administered percutaneously once daily for a 6-hour exposure period on day 6 through 15 of presumed gestation. The dosage volume was 1 mL/kg bw based on the day 6 of presumed gestation body weight of each dam and applied to a surface area of approximately 20 cm2on each rat's back; Groups I and II were given the Vehicle (reverse osmosis membrane processed deionised water). Groups III and IV were given the Blank (a formulation containing solvents and surfactants at equivalent concentrations to those in the 10 mg/kg bw/day dose group). Groups V through X were given 2, 5, 10, 15, 20 and 75 mg/kg bw/day dosages of the test substance. Test dosage solution was applied with a blunt-tipped disposable syringe. General health and viability were noted twice daily. Skin reaction to test article and abortion incidence were recorded daily (prior to dosing) from day 6 to day 15 of presumed gestation, and from day 16 to day 20 postdosage period. Body weights were recorded at day 0 and days 6 through 20. Food consumption was recorded between days 0 and 6 of presumed gestation and daily on days 6 through 20. All rats were sacrificed on day 20. Thoracic and abdominal cavities were examined for gross lesions, corpora lutea in each ovary were counted, uterus was excised and weighed and examined for pregnancy, number and placement of implantations, early and late resorptions and live and dead fetuses. Each fetus was removed from uterus and examined to identify sex and gross external alterations. Live fetuses were sacrificed and examined for soft tissue alterations, and also stained with alizarin red S to facilitate for skeletal alterations. Dosing solutions (prepared daily) were analysed and found to be accurate, except for the 2 mg/mL group where intended concentration was only 62% of theorical concentration.

Pregnancy incidences were comparable in all dosage groups. All maternal, litter and fetal evaluations were based on 21 to 24 pregnant rats with live day-20 of gestation Caesarean-delivered fetuses, with the exception of one group (15 mg/kg bw/day), in which there were only 18 day-20 of gestation litters.

Significant numbers of rats in the two groups applied the Blank had slight erythema, and significant numbers of rats in the 75 mg/kg bw/day dosage group had slight erythema, moderate erythema and slight fissuring at the application site. Percutaneous application to the dams in this study at dosages as high as 75 mg/kg bw/day did not result in death or clinical (other than the skin reactions noted above) or necropsy observations.

Significant, dosage-dependent inhibitory effects on average maternal body weight gains occurred for dams in the 20 and 75 mg/kg bw/day dosage groups, as compared with values for groups applied the vehicle. Average maternal feed consumption values were significantly decreased for the dams in the 15, 20 and 75 mg/kg bw/day dosage groups during various intervals between days 6 and 12 of gestation. Between days 12 and 16 of gestation and during the postdosage period, significant increases in relative feed consumption values occurred for the 75 mg/kg bw/day dosage group, a rebound phenomenon. This rebound phenomenon persisted for the 75 mg/kg bw/day dosage group during the postdosage period, when significant increases in relative maternal feed consumption also occurred.

Fetal body weights tended to be decreased for the 75 mg/kg bw/day dosage group, as compared with the values for the groups applied the vehicle. No other observation at Caesarean-sectioning of the dams was attributed to treatment. Average values for corpora lutea, implantations, litter sizes, live and dead fetuses, and early and late resorptions were comparable among the groups and did not significantly differ. Similarly, fetal sex ratios and viability indices were not affected by percutaneous application at dosages as high as 75 mg/kg bw/day.

As compared with the vehicle control group values, the maternally toxic 20 and 75 mg/kg bw/day dosages produced significant, dosage-dependent increases in the incidence of 14th thoracic ribs (with related significant changes in the thoracic and lumbar vertebral incidences). At 15 mg/kg bw/day there were very slight, non-statistically significant, increases in the number of fetuses with extra ribs. No other alterations (malformations or variations) in fetal external, soft tissue or skeletal morphology were considered related to percutaneous administration of the test substance to the dams as dosages as high as 75 mg/kg bw/day. No other fetal alterations occurred at incidences that were dosage-dependent, biologically remarkable and significantly increased, as compared with the values for the vehicle control groups.

Based on the data in this study, the maternal NOAEL for general toxicity was 10 mg/kg bw/day. Dosages of 15 mg/kg bw/day and above significantly inhibited relative maternal feed consumption values during the first two-thirds of the dosage period. Dosages of 20 and 75 mg/kg bw/day significantly inhibited average maternal body weight gains during the dosage period.

The developmental (embryo-fetal toxicity and teratogenicity) NOAEL was 10 mg/kg bw/day. At 15 mg/kg bw/day, non-significantly increases in the number of fetuses with extra ribs were noted. The maternally toxic 20 and 75 mg/kg bw/day dosages of the test substance produced significant, dosage-dependent increases in the incidence of 14th thoracic ribs.

 

 

In addition to the data obtained for the target substance, one GLP compliant study (M-227377-01-1, 1988) was performed in rats with the source substance (CAS 1689-84-5) according to U.S. EPA Pesticide Assessment Guideline; Subdivision F, Hazard Evaluation: Human and Domestic Animals, November 1984, 83-3, Teratology Study. 23 pregnant rats per group were exposed percutaneously to the source substance at dose levels of 5, 10, 50 and 100 mg/kg bw/day as solutions in reverse osmosis membrane processed deionised water with sodium hydroxide (50 mg/mL) and triethylene glycol (20% of the total volume). All dosages were given at a dosage volume of 2 mL/kg bw (subsequently adjusted for body weight changes) applied to the shaved skin surface area of approximately 35 cm2on each rat's back. The exposure lasted 6 h/day and was performed daily from Day 6 - 15 of gestation. A concurrent vehicle control group was included. Day 0 of presumed gestation was defined as the day spermatozoa were identified in a smear of the vaginal contents or a copulatory plug was present in situ. All surviving animals were sacrificed on gestation Day 20. General health and viability were noted twice daily. Skin reaction to test article and abortion incidence were recorded daily (prior to dosing) from day 6 to day 15 of presumed gestation, and from day 16 to day 20 post dosage period. Body weights were recorded at day 0 and days 6 through 20. Food consumption was recorded between days 0 and 6 of presumed gestation and daily on days 6 through 20. With the exception of one rat from the 10 mg/kg bw/day dose group that was sacrificed on day 18 of presumed gestation after it naturally delivered its litter, all rats were sacrificed on day 20. Thoracic and abdominal cavities were examined for gross lesions, corpora lutea in each ovary were counted, uterus was excised and weighed and examined for pregnancy, number and placement of implantations, early and late resorptions and live and dead fetuses. Each fetus was removed from uterus and examined to identify sex and gross external alterations. Live fetuses were sacrificed and examined for soft tissue alterations, and also stained with alizarin red S for examination of skeletal alterations. Because increased rib incidence was the lowest developmental effect identified in oral developmental toxicity studies in rats, the rib incidence was independently identified a second time for each fetus by a second investigator.

Data of maternal body weight, gravid uterine weight, food consumption, litter average for percent male fetuses, fetal body weights, fetal ossification sites and fetal alterations were tested for homogeneity of variance by Bartlett's test or Analysis of Variance, where appropriate. Where Analysis of Variance was significant, Dunnett's test was used to identify the statistical significance difference between individual groups. If the data was found to be non-parametric, the data was subjected to Kruskal-Wallis Test or Fisher's Exact Test, where appropriate. Dunn's Test was used to identify the statistical significance difference between individual groups.

No death occurred during the course of the study and no adverse skin reactions or clinical signs related to treatment were observed. Localized alopecia was a common observation but it occurred in pregnant rats across all the treatment groups including control. There were no other necropsy observations. Maternal body weight gain (mean) was reduced between gestation Day 6 and 12 in the groups receiving 50 and 100 mg/kg bw/day (22 and 38% in the 50 and 100 mg/kg bw/day group, respectively). Between days 12 and 16 of gestation there was a rebound effect in these 2 dosage groups in which body weight gain increased (p<0.05), but the overall body weight gains were only 90% of the control group for the entire dosage period (Days 6 to 16 of gestation). Average maternal food consumption values for the entire dosage period (days 6 to 16 of gestation) did not markedly differ among the groups. However, for the 100 mg/kg bw/day group, maternal feed consumption was reduced between Days 6 and 9 of gestation but a subsequent rebound effect was observed.

The number of pregnancies, average values for corpora lutea, implantations, litter sizes, live and dead fetuses, and early and late resorptions were comparable among the groups, with no statistically significant differences. Fetal sex ratio, body weights and viability indices were found to be unaffected by treatment in any of the dose groups. No gross fetal malformations were noted.

In the 50 and 100 mg/kg bw/day group, a statistically significant (p<0.01) increase in a variation in rib ossification (14th ribs) in the fetuses occurred. This was related to statistically significant changes in thoracic and lumbar vertebral incidences, as compared to the control group, but this variation is common in this rat strain. The finding of higher incidences of the supernumerary 14th rib is coherent with the observation in the previously reported oral teratogenicity study in the rat (M-184740-01-1).

No other variations (external alterations such as exencephaly, open eyes, cleft palate, facial cleft, soft tissue alterations such as close-set kidneys, skeletal alterations such as cranio-facial malformations e.g. incomplete ossified palate, no ossification in the nasal bones) observed were attributed to the test article, as such incidences were incidental and occurred across all the groups including control.

In conclusion, the maternal NOAEL for dermal application of the source substance was 10 mg/kg bw/day based on altered body weight, body weight gain and feed consumption at higher dose levels. The developmental (embryo-fetal toxicity and teratogenicity) NOAEL was set at 10 mg/kg bw/day based on increases in the incidences of the 14th ribs in fetuses of the 50 and 100 mg/kg bw/day group. However, it is inconclusive if the embryo-fetotoxic effects observed in this study are independent of maternal toxicity, since they occur at the same dose levels.

 

Rabbits 

The target substance (CAS 1689-99-2) was administered percutaneously once daily for a 6-hour exposure period to artificially inseminated rabbits on days 6 through 18 of presumed gestation (according to GLP and US EPA Pesticide Assessment Guidelines. Subdivision F, 83-3;M-227408-01-1). Groups I and II were given the vehicle. Groups III and IV were given the Blank (a formulation containing solvents and surfactants at equivalent concentrations to those in the 10 mg/kg/d dose group). Groups V through X were given 5, 10, 15, 20, 40 and 80 mg/kg bw/day dosages of the test substance. A dosage volume of 1 mL/kg was used and adjusted daily on the basis of body weight and applied to a surface area of approximately 150 cm2on each rabbit's back. Each rabbit was observed daily during the dosage and postdosage periods for evidence of skin reactions and other clinical signs of test substance effects, including deaths, abortions, premature deliveries, body weight changes and feed consumption decrements. Does that died, aborted, prematurely delivered or were sacrificed for humane reasons were necropsied on the day the event occurred and examined for pregnancy and live and dead implantations; maternal tissues with gross lesions present were retained in neutral buffered 10% formalin. Conceptuses from these does were examined to the extent possible. On day 29 of presumed gestation, all other rabbits were sacrificed, Caesarean-sectioned, and examined for gross lesions. The intact uterus of each rabbit was removed, weighed, and subsequently examined for number and placement of implantations, early and late resorptions, and live and dead fetuses. Each ovary was examined for the number of corpora lutea. Fetuses were subsequently weighed and examined for external, soft tissue and skeletal alterations. Dosing solutions (prepared daily) were analysed and found to be accurate.

No deaths, abortions or premature deliveries were caused by percutaneous administration at dosages as high as 80 mg/kg bw/day. The incidences of these events were not dosage-dependent. One doe given the vehicle died. One doe given the vehicle and 1 doe given the Blank aborted. Another doe given the Blank had a litter consisting of 8 early resorptions, an observation that is sometimes interpreted as an abortion. One doe given the vehicle and 1 doe given the 15 mg/kg bw/day dosage of the test substance prematurely delivered. One nonpregnant doe given the 40 mg/kg bw/day dosage of the test substance was sacrificed because it had a broken back. All other does in this study survived to day 29 of presumed gestation and had live litters.

The vehicle alone caused minimal skin reactions (1 of a total of 40 does in Groups I and II had grade 1 erythema noted once). The blank alone caused significant numbers of rabbits in Groups III and/or IV to have grade 1 or 2 erythema, grade 1 fissuring and/or grade 1 desquamation and a few rabbits to have grade 3 or 4 erythema, grade 2 fissuring and grade 2 desquamation.

Dosages of 5 to 80 mg/kg bw/day caused dosage-dependent increases in skin reactions (significant numbers of rabbits in each group given the test substance had at least grade 2 erythema). The 5 mg/kg bw/day dosage of the test substance was less irritating than the Blank alone, and the 40 and 80 mg/kg bw/day dosages were more irritating than the Blank alone. No other dosage-dependent or statistically significant clinical or necropsy observations occurred.

The 40 and 80 mg/kg bw/day dosages caused biologically important reductions in maternal body weight gains during the dosage period (calculated as days 6 to 19 of gestation), as compared with values for groups given the vehicle (Groups I and II) and the Blank (Groups III and IV). During the postdosage period (calculated as days 19 to 29 of gestation), the 40 and 80 mg/kg bw/day dosage groups had increased body weight gains, as compared with groups given the vehicle or the Blank. These observations were considered to be rebound phenomena and were most obvious when comparisons were made with the values for the vehicle control groups (Groups I and II).

There were no dosage-dependent effects on average gravid uterine weights. Reflecting the increased maternal body weight gains that occurred for the 40 and 80 mg/kg bw/day dosage groups during the postdosage period, there were no dosage-dependent effects on average maternal body weight gains between days 6 and 29 of gestation. Percutaneous application of the test substance at dosages as high as 20 mg/kg bw/day did not cause biologically important differences in maternal body weight gains or body weights during the dosage or postdosage periods.

The 80 mg/kg bw/day dosage caused biologically important reductions in absolute and relative maternal feed consumption values, as compared with values for Groups I and II or Groups III and IV during the dosage period. During the postdosage period, the 40 and 80 mg/kg bw/day dosage groups had increased or significantly increased absolute and relative maternal feed consumption values, as compared with values for Groups I and II, and increased absolute and relative maternal feed consumption values, as compared with the Groups III and IV. These observations were considered to be rebound phenomena.

No observation at Caesarean-sectioning of the does was attributed to the test substance. Average values for corpora lutea, implantations, litter sizes, live and dead fetuses, and early and late resorptions were comparable in the 10 groups; differences were not significant. Similarly, fetal sex ratios, body weights and viability indices were not significantly affected by dosages as high as 80 mg/kg bw/day.

Administration of the test substance to the does at dosage as high as 80 mg/kg bw/day did not result in fetal external, soft tissue or skeletal alterations (malformations or variations). The incidences of all fetal alterations were either not statistically significant, as compared with values for the vehicle control groups, or if statistically significant, were not dosage-dependent.

Based on the data in this study, the maternal NOAEL for general, systemic toxicity was 20 mg/kg bw/day. Dosages of 40 and 80 mg/kg bw/day reduced maternal body weight gains, and the 80 mg/kg bw/day dosage reduced absolute and relative maternal feed consumption values. All dosages of the test substance produced skin irritation; observations for the 40 and 80 mg/kg bw/day dosage group were more severe than for the group given the Blank.

The developmental NOAEL≥80 mg/kg bw/day. There were no adverse effects on embryo-fetal development at the highest dosage tested (80 mg/kg bw/day).

 

Further, one additional GLP compliant study on developmental toxicity following dermal exposure of the source substance (CAS 1689-84-5) is available in rabbits according to U.S. EPA Pesticide Assessment Guideline; Subdivision F, Hazard Evaluation: Human and Domestic Animals, November 1984, 83-3, Teratology Study (M-227401-01-2, 1988). 20 presumed pregnant New Zealand White rabbits per group were exposed to dose levels of 10, 50 and 150 mg/kg bw/day percutaneously. All dosages were given at a dosage volume of 2 mL/kg bw (subsequently adjusted for body weight changes) applied to the shaved skin surface area of approximately 150 cm2on each rabbit's back. Dosages were given to the rabbits as solutions in reverse osmosis membrane processed deionised water with sodium hydroxide (50 mg/mL) and triethylene glycol (20% of the total volume). Test dosage solution was applied with a blunt-tipped disposable syringe. The exposure lasted 6 h/day and was performed daily from Day 6 - 18 of gestation. Day 0 of presumed gestation was defined as the day insemination occurred. All surviving animals were sacrificed on gestation Day 29. The animals were observed for a period of 29 days. General health and viability were noted twice daily. Skin reaction to test article and abortion incidence were recorded daily (prior to dosing) from day 6 to day 18 of presumed gestation, and from day 19 to day 29 post-dosage period. Body weights were recorded at day 0 and days 6 through 29. Food consumption was recorded daily. Necropsy observations and uterine contents were recorded for rabbits that aborted or failed in health. All surviving animals on day 29 of presumed gestation were sacrificed and necropsy performed. Thoracic and abdominal cavities were examined for gross lesions, corpora lutea in each ovary were counted, uterus was excised and weighed and examined for pregnancy, number and placement of implantations, early and late resorptions and live and dead fetuses. Each fetus was removed from uterus and examined for gross external alterations. Live fetuses were sacrificed and examined for visceral alterations and brain abnormalities. The fetuses were then eviscerated and stained with alizarin red S and evaluated for skeletal alterations. Data of maternal body weight, gravid uterine weight, food consumption, litter average for percent male fetuses, fetal body weights, fetal ossification sites and fetal alterations were tested for homogeneity of variance by Bartlett's test or Analysis of Variance, where appropriate. Where Analysis of Variance was significant, Dunnett's test was used to identify the statistical significant difference between individual groups. If the data was found to be non-parametric, the data was subjected to Kruskal-Wallis Test or Fisher's Exact Test, where appropriate. Dunn's Test was used to identify the statistical significant difference between individual groups.

Dose analyses revealed a possible dosing error in certain animals on day 6 and 7 of presumed gestation. Despite this error the interpretation and validity of the study was not affected and the same conclusions are reached with the exclusion of the data of the does that were possibly given incorrect dosages. Other dosing solutions (prepared daily) were analysed and were found to be accurate.

One animal of the high dose group was sacrificed on Day 25 of gestation because of moribund condition. It could not be determined if the death was treatment-related, since tricobezoar was present in the doe's stomach at necropsy. Percutaneous administration of the test substance to the does at dosages up to 150 mg/kg bw/day did not cause treatment-related abortions. Abortion occurred only in one control doe and one doe of the 10 mg/kg bw/day group. Due to missing dose-response relationship, it was therefore considered incidental. The incidences of does with skin reactions or clinical or necropsy observations did not significantly differ among the groups. Maternal body weight gain was reduced throughout the study in the high dose group. Food consumption was reduced in the 50 and 150 mg/kg bw/day group between Days 12 and 13, and Days 13 and 14 of gestation.

Average values for corpora lutea, implantations, litter sizes, live and dead fetuses and early and late resorptions were comparable between the treatment and vehicle control groups. Similarly, fetal sex ratios, body weights and viability indices were not significantly affected by the test article at dosages as high as 150 mg/kg bw/day. There was no evidence of treatment-related effects on fetal alterations (malformations or variations). However, there was a statistically significant (p<0.01) increase in the litter incidence of a variation in lung morphology (agenesis of the intermediate lobe) from the 150 mg/kg bw/day dose group, but this was within the range of historical control data and is therefore not considered as treatment related. None of the observed alterations, such as central nervous system alterations, slight dilation of lateral ventricles of the brain, irregular ossification of the nasal area of skull, irregular ossification of the frontals of the skull, were attributed to treatment-related effects.

Based on the data in this study the maternal no-observable adverse effect level for the source substance is between 10 mg/kg bw/day and 50 mg/kg bw/day. Dosages of 50 and 150 mg/kg bw/day significantly (p≤0.05) decreased average maternal feed consumption. The 150 mg/kg bw/day dosage resulted in significant (p≤0.05) decreases in average maternal body weight gains between days 6 to 29 (day 29 of gestation body weight corrected for the weight of the gravid uterus). One doe given the 150 mg/kg bw/day dosage of the test substance was moribund sacrificed as the result of observations attributed to this dosage another doe had ulcerated areas in the gall bladder. The developmental (embryo-fetal toxicity and teratogenicity) no-observable adverse effect level is greater than 150 mg/kg bw/day. If the significant (p≤0.01) increase in agenesis of the intermediate lobe of the lungs is considered an effect of this maternally toxic dosage of the test substance, the developmental NOAEL is between 50 and 150 mg/kg bw/day. It is concluded that percutaneous application may cause embryo-fetotoxicity only at a dosage that also causes obvious conventional signs of maternal toxicity (significant inhibitory effects on body weight gain and feed consumption).

 

Mechanistic investigations

An additional mechanistic study was conducted in pregnant rats to further assess maternal toxicity in rats following oral dosing of the source substance.

In this mechanistic study, the test substance was administered daily by oral gavage to groups of 10 female pregnant Sprague-Dawley rats (M-509605-01-1, 2015). The study was not performed under GLP conditions but according to US EPA OCSPP 870.SUPP guideline. Animals received the source substance (CAS 1689-84-5) from Gestation Day (GD) 6 to 21 at dose levels of 5, 12.5 and 30 mg/kg bw/day. A similarly constituted group received the vehicle (0.5% aqueous methylcellulose 400 + 0.5% acetic acid) and served as a control. Animals were observed at least once daily for mortality and clinical signs. Body weight was recorded at regular intervals and food consumption was calculated daily. All animals were necropsied on GD 21, gravid uterine weight was recorded and the liver was weighed. Liver samples were taken for histological and electron microscopy examinations, and for gene transcript analyses by quantitative Polymerase Chain Reaction (qPCR).

There was no mortality and no treatment related clinical signs up the highest dose level of 30 mg/kg bw/day. Transient body weight loss and decreases in overall body weight and body weight gain were observed at 30 mg/kg bw/day. Dose related increases in liver weight and enlarged liver observed macroscopically at 12.5 and 30 mg/kg bw/day correlated with evidence of centrilobular hypertrophy in these 2 groups. A slight increase in glycogen accumulation in the hepatocytes without any alteration of organelles in hepatocytes and non hepatocytic cells was observed at 30 mg/kg bw/day. This finding is considered not to be of toxicological relevance but associated with a consequence of the non-fasting status of the pregnant animals prior to sacrifice and a slight increase of food intake at this dose at the end of the study. The qPCR results indicated that the great majority of the gene transcripts selected and involved in regulating oxidative phosphorylation or cell ATP content were deregulated in a dose dependent manner in the liver of treated female rats at 12.5 and 30 mg/kg bw/day. Minor changes in some gene transcripts at 5 mg/kg bw/day were considered not to be toxicologically relevant in the absence of changes in the liver.

The NOAEL was considered to be 5 mg/kg bw/day.

Conclusion on developmental toxicity

In a series of studies performed in the rat and the rabbit, either by the oral or dermal route of administration, developmental toxicity induced by the source substance was investigated.

Briefly, oral administration of the source substance to pregnant rats at 0, 5, 15 or 35 mg/kg bw/day produced maternal toxicity in the form of reduced bodyweight gain (mid and high dose groups) and mortality (high dose) (M-184740 -01-1). There was no evidence of teratogenicity. Reduced fetal weight, increased pre-implantation losses and increased late uterine deaths observed at 35 mg/kg bw/day may have been related to a direct effect upon the embryo/fetus, although maternal toxicity could well have been a contributory factor. The non-statistically significant but dose related increase in minor fetal anomalies and the statistically significant dose related increase in the supernumerary 14th rib variant may both be regarded as being compound related. Although the applicant considered that the lowest dose level (5 mg/kg bw/day) was a NOAEL for both maternal and developmental toxicity, the EU review concluded that the NOAEL was < 5 mg/kg bw/day based on slight non-statistically significant effects on maternal bodyweight and the incidence of 14th supernumerary ribs. An overall developmental NOAEL of 4 mg/kg bw/day was established based on increased malformations (anophthalmia and microphthalmia, diaphragmatic hernia, narrowing of the aortic arch and fusion of ribs associated with scoliosis) and supernumerary ribs in a separate study (M-344549-01-2).

Oral administration to pregnant rabbits at 0, 15, 30 or 60 mg/kg bw/day produced maternal toxicity in the form of reduced bodyweight gain and food consumption in the mid and high dose groups(M-184736-01-2). Significant increases in major malformations indicative of teratogenicity (anophthalmia and microphthalmia, hydrocephalus and ossification effects) were observed at the highest dose only. Embryo-fetotoxicity at this dose level was characterised by increased post implantation losses, increased supernumerary rib variants and reduced fetal weight. No significant developmental effects were observed at lower dose levels. According to the EU review, the NOAEL for maternal toxicity was < 15 mg/kg bw/day based on a slight non-statistically significant decrease in mean body weight gain at this dose level. The developmental NOAEL was < 15 mg/kg bw/day based on a non statistically significant increase in the incidence of supernumerary ribs at this dose level. As outlined in this dossier, the notifer considers that a maternal NOAEL of 15 mg/kg bw/day and a developmental NOAEL of 30 mg/kg bw/day can be established in this study.

Administration of the source substance by the dermal route to pregnant rats resulted in maternal toxicity (reduced bodyweight) and evidence of embryo-fetotoxicity (increased incidence of 14th supernumerary ribs) at maternally toxic doses (M-227377-01-1). There was no evidence of teratogenicity. The maternal and developmental NOAELs were 10 mg/kg bw/day. Corresponding studies in pregnant rabbits produced maternal toxicity (reduced bodyweight) but no adverse developmental effects up to the highest doses tested (M-227401-01-1). The maternal NOAEL was 10 -50 mg/kg bw/day, respectively, whilst the developmental NOAEL was > 150 mg/kg bw/day, respectively.

Based on the obtained data, the source substance and its ester derivatives, including the target substance are classified Repr. 2, H316d Suspected of damaging the unborn child according to Annex VI of Regulation (EC) No 1272/2008 (CLP Regulation).

Discussion of developmental toxicity studies in relation to classification and labelling

A study designed to investigate the rat reproductive cycle through two successive generations showed that the source substance had no adverse effects on mating performance, sexual function, fertility, lactation or the outcomes of the pregnancies. Slight delays in neonatal growth were seen at the higher doses but they did not affect the animals’ maturity to adulthood and crucially no physical evidence for such a slight effect was present at further physical examination or at necropsy. The evidence suggests that there were no demonstrable physical anomalies present that affected the offspring to successfully reproduce a second generation. The data clearly suggested that, at the treatment levels studied, the source substance did not adversely affect the development of the offspring treated continuously in utero, via lactation and later via their diet. Thus confirming, in this study, that the source substance was without adverse effect on the complete reproductive cycle in the rat. Additionally, further work on the male rat corroborated these findings in confirming that the source substance had no adverse effects on spermatogenesis and consequent fertility. In consideration of the classification and labelling criteria (EU CLP regulation 1272/2008, 3.7); no adverse effects on sexual function, fertility, development of the conceptus, lactation or the development of the offspring clearly indicate that classification is unwarranted for the source substance and its ester derivatives.

In a series of developmental studies in the rat, either by the oral or dermal route of administration, a number of effects on the development of the foetus was apparent at doses (via the oral route ) that induced clear and obvious maternal toxicity. In the rat, the findings associated with maternal toxicity included increases in supernumerary ribs. Such findings may be considered to be reversible skeletal variants (as part of a normal background of natural variants, <1% to >30% - Chernoff & Rogers, 2004) and may be considered to be of low concern (as described in Moore et al, 2013) in relation to classification. Indeed the findings from the multigeneration study corroborate this as littering parameters were not adversely affected. Furthermore, there appears to be no consensus regarding non-specificity and relationship to maternal toxicity for supernumerary ribs in rats, although increased incidences have been shown to occur in the presence of maternal toxicity with many different substances of different classes acting via different modes of action (Paumgartten, 2010) or indeed they may be stress related (EU CLP regulation 1272/2008, 3.7.24). Such small or rudimentary ribs are considered to be ossification sites that are later absorbed into the adjacent vertebrae, and probably represent little more than a natural developmental variation (Chernoff & Rogers, 2004). Furthermore,it is widely believed that maternal toxicity may, depending on severity, influence development via non-specific secondary mechanisms, producing effects such as depressed foetal weight, retarded ossification, and possibly resorptions and certain malformations in some strains of certain species (Moore et al, 2013;EU CLP regulation 1272/2008, UN GHS, 2013).In the presence of sometimes marked maternal toxicity the toxicological significance of the supernumerary ribs, described in the studies presented in this dossier, can be considered doubtful as a specific development deficit.

In a second species developmental toxicity study, conducted in the rabbit, via the oral route, administration of the source substance did suggest some evidence of foetal abnormalities at treatment levels where marked/severe maternal toxicity was evident. At the highest dosage there were marked and persistent decreases in maternal food intake and body weight gain. It was at the highest dosage tested (60 mg/kg bw/day, considerably higher than the top dosage tested in rats) that the foetal abnormalities were seen without a dosage related trend. Foetal abnormalities in the presence of such marked/severe maternal toxicity need not necessarily be discounted, but such foetal effects were probably as a result of non-specific secondary mechanisms rather than a direct primary effect. In such circumstances it is probable that in utero effects could be attributed to the physiologically compromised pregnant female (e.g. hepatotoxicity) – in particular during key phases of organogenesis. Two further developmental studies were conducted in the rabbit with the source substance and an ester derivative (see analogue justification), via the dermal route at a much higher top dosage level than dosed via the oral route. In neither study were foetal abnormalities seen, nor were there any marked adverse effects on maternal condition as seen previously. This illustrates that the route of exposure probably affected the rate systemic availability of the test material and peak blood levels. Dermal penetration may be in the region of 5% to 10% for the esters (Dermal absorption study summaries, please refer to the data provided under “Toxicokinetics”) and the dermal acute or repeat dermal dose toxicity studies on this substance clearly suggested that systemic availability of the test substance was limited compared to that via the oral route. Oral doses to rabbits (around 3 to 4 mL dosing suspension per gavage dose) were probably rapidly absorbed and at a high dosage (60 mg/kg bw/day) resulted in the marked maternal effects seen from early in the dosing period of the rabbits (organogenesis). It is probable that the rapid and acute systemic availability of the source substance, at this dose level, was a key factor in inducing the marked maternal toxicity seen. This high dosage could represent a maximum tolerated dose level in this species and the lack of dosage related effects further brings into question using such finding, at 60 mg/kg bw/day, to define an unequivocal developmental toxicity risk. As previously mentioned, the high oral dosage used for the rabbits was significantly higher than that used in the definitive study in the rat. This further supports and suggests that the adverse maternal condition seen in the rabbit cannot be dissociated from the foetal abnormalities seen at the highest dose tested.

Two MoA studies were conducted in female rats (non-pregnant (M-509609-01-1, 2015) and pregnant (M-509605-01-1, 2015 (as summarized above)), at dosages relevant to the developmental and reproduction studies and showing some comparable maternal toxicity, showed that there were significant effects on the livers of the rats. The qPCR results indicated that the majority of the gene transcripts investigated, and involved in regulating oxidative phosphorylation or cell ATP content, were deregulated in a dose dependent manner at 12.5 and 30 mg/kg bw/day. As shown in the mammalian toxicity studies for the source substance the liver was the major target organ – with effects seen at the dosages used in the developmental studies. Such effects as demonstrated in the MoA studies would suggest that changes in oxidative phosphorylation in the liver most probably contributed to the adverse maternal toxicity that preceded the foetal effects seen in the developmental studies. From the overall toxicological evidence, seen for the source substance, it is likely that the effects on the maternal liver were key contributory factors in the foetal effects seen in the developmental studies.

In summary, the source substance is classified as a suspected reproductive and developmental toxicant (Category 2, H361d) following the recent harmonisation to CLP.

The comprehensive reproductive and developmental toxicity dataset has been evaluated against the current CLP criteria and guidance, using a stepwise, systematic, weight and strength of the evidence approach including the most up-to-date scientific knowledge.

The outcome of the expert evaluation is that while there is sufficient evidence to support that the source substance may meet the CLP criteria for classification as a suspected developmental toxicant (Category 2, H361d, ‘May damage the unborn child’), it does not meet the CLP criteria for classification as a presumed or known reproductive and developmental toxicant (Category 1) or the criteria for lactation effects.

 

References not included in IUC:

Detailed information on references not included in IUC are available in the CSR and in chapter 13.

Mode of Action Analysis / Human Relevance Framework

Please refer to the endpoint summary. 

Justification for classification or non-classification

The available data on reproductive toxicity meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore sufficient for classification of the test substance for reproductive toxicity category 2 (Repr. 2; H361d).

The substance is listed in Annex VI of Regulation (EC) 1272/2008 (Index No. 608-017-00-0) as a reproductive toxicant category 2 (Repr. 2; H361d).