Bis-O-(benzylidene)-D-glucitol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The reproductive toxicity of the substance (EC 251-136-4) has been assessed by reading across the results of studies conducted on two structurally similar analogue substances, which are detailed below.

Source Substance; Gel All DX (EC-413 -110 -2): Reproduction/Developmental Toxicity Screening Test (OECD 421):

The purpose of the study was to generate preliminary information concerning the effects of Gel All DX on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

 

The test item was administered once daily orally (by gavage) throughout pre-pairing, pairing and after pairing periods for a total of 46 days in males and pre-pairing, pairing, gestation and lactation periods for at least 40 days in females, up to the day before scheduled necropsy.

 

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (olive oil).

Parent Animals

Mortality/Viability

No test item related deaths were noted during the study.

 

One male given 1000 mg/kg body weight/day was found dead on day 12 of the after pairing period. The cause of this death was unclear and, due to an isolated occurrence, unlikely to be related to toxicity of the test item.

Clinical Signs

There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to the administration of the test item.

Food Consumption

There was no effect of the test test item on the group mean amount of food consumed by males or females at any dose level over the course of this study, when compared to control animals.

Body Weights

Group mean body weight and body weight gain for males and females were considered to be unaffected by administration of the test item over the duration of the study.

Reproduction and Breeding Data

Fertility, time course of mating, duration of gestation,corpora luteacount, number of implantation sites, post-implantation loss, liter size and post natal loss were unaffected by administration of the test item at any dose level.

Organs Weights

No changes in absolute testes and epididymides weights or weights of these organs relative to body weight were noted at any dose level.

Macroscopic/Microscopic Findings

No test item-related macroscopical or microscopical findings were recorded at any dose level.

Litter Data - F1 Pups

Findings at First Litter Check and During Lactation

No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

 

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

Pup Weights to Day 4Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

No test item-related findings were noted in pups at necropsy at any dose level.

Conclusion

Based on these results, NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Source Substance; Gel All MD (EC 402-950-5): OECD 415 One-Generation Reproduction Toxicity Study:

The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat to assess prenatal and postnatal development, and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).

The administration of the test material by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of

the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13-Jun-2013 to 01-Nov-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Animals: Rat, RccHanTM: WIST(SPF)
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Harlan Laboratories, B.V., Netherlands
Number of Animals: 48 males: 12 per group
48 females: 12 per group
Age (at Start of Treatment): Approximately 11 weeks
Body Weight Range (at Start of Treatment): Males 329 to 382 g
Females 185 to 236 g
Identification: Cage card and individual animal number (ear tattoo).
Randomization: Performed after at least three days of acclimatization using a computer-generated random algorithm. Body weights (recorded on the day of allocation) were taken into consideration in order to ensure similar mean body weights in all groups.
Acclimatization: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

HUSBANDRY:
Conditions:
Standard laboratory conditions. Air-conditioned with 10 - 15 air changes per hour, continuously monitored environmental conditions (temp. range: 18 ± 3 °C from 13-Jun-2013 to 26-Jun-2013 and 22 ± 3 °C from 27-Jun-20113 onwards; relative humidity range: 30 - 70%). Values outside of these ranges occasionally occurred, usually following room cleaning, which was considered not to have any influence on the study. The light cycle was set to 12-hour fluorescent light / 12-hour dark cycle

Accommodation:
Individually in Makrolon type-3 cages with wire mesh tops and sterilized stan¬dard softwood bedding with paper enrichment. During the pre-pairing period, cages with males were interspersed amongst those holding females to promote the development of regular estrus cycles.

Diet:
Pelleted standard Harlan Teklad 2018C rodent maintenance diet was available ad libitum.

Water:
Community tap-water was available ad libitum in water bottles.

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly using the test item as supplied.

Separate formulations were prepared for each concentration.

For dose formulations in groups 2 (100 mg/kg bw/day) and 3 (300 mg/kg/bw/day), Gel All DX was weighed into a glass beaker on a tared precision balance and approximately 80% of the vehicle was added (w/v). Using an appropriate homogenizer, a homogeneous suspension was prepared. Having obtained a homogeneous mixture, the remaining vehicle was added.

For dose formulation in group 4 (1000 mg/kg bw/day), Gel All DX was weighed into a glass beaker on a tared precision balance and approximately 60% of the vehicle was added to another glass beaker. The test item was added successively to the vehicle under constant mixing. Having obtained a homogeneous mixture containing entire amount of the test item, the remaining vehicle was added.

Homogeneity of the test item in the vehicle will be maintained during the daily administration period using a magnetic stirrer.

STORAGE OF DOSE FORMULATIONS:
Dose formulations were stored in refrigerator (5 ± 3 °C) in glass beakers.

Based upon the results of stability analyses performed within 14-Day Dose Range-Finding Study in the Han Wistar Rat, dose formulations were stable for at least 8 days if stored in refrigerator (5 ± 3 °C).

TREATMENT:
Method: Oral, by gavage
Rationale for Method: Administration by gavage is a common and accepted route of exposure for studies of this type.
Frequency of Administration: Daily, at approximately 24 hour intervals.

Dose Levels:
Group 1: 0 mg/kg/day (control group)
Group 2: 100 mg/kg/day
Group 3: 300 mg/kg/day
Group 4: 1000 mg/kg/day

Dose Volume: 5 mL/kg body weight

Dose Concentrations:
Group 1: 0 mg/mL
Group 2: 20mg/mL
Group 3: 60 mg/mL
Group 4: 200 mg/mL

Duration of Acclimatization Period: Minimum 5 days

Duration of Treatment Period:
Males: 46 days
Females: Approximately 7 weeks

Details on mating procedure:

During the pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed.
The females were removed and housed individually if:

- the daily vaginal smear was sperm positive, or
- a copulation plug was observed.

The day on which a positive mating was determined (copulation plug or sperm) was designated day 0 post coitum. No sperm or copulation plug was noted in two females, no. 84 in group 3 and no. 86 in group 4, during the first pairing period. However, as a body weight gain typical for pregnancy was observed in these females, it was considered that mating of these females was overlooked and therefore they were not paired with a second partner, but instead transferred to gestation after 13 days of the pairing period.

All dams were allowed to give birth and rear their litters (F1 pups) up to day 4 post partum. Day 0 post partum was designated as the day on which a female had delivered all her pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On the first treatment day samples from the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of concentration and homogeneity. To confirm the stability (8 days at 5 ± 3 °C) samples of about 1 g of each concentration were taken from the middle of each aliquot used on day 7 of the treatment. During the last week before first planned necropsies, samples were taken from the middle to confirm concentration. The aliquots for analysis of dose formulations were delivered to the analytical laboratory immediately after collection at ambient temperature and stored there at -20 ± 5 °C until analysis.

The samples were analyzed by HPLC coupled to an UV detector . The test item was used as the analytical standard.

Duplicates were taken of all samples.

In blank sample chromatograms no peak appeared at the retention time of Gel All DX and, therefore, the absence of the test item in the vehicle control samples (olive oil) was confirmed. The Gel All DX concentrations in the dose formulations ranged from 89.5% to 102.6% with reference to the nominal and were within the accepted range of ±20%.

The homogeneous distribution of Gel All DX in the preparations was approved because single results found did not deviate more than 0.9% from the corresponding mean and met the specified acceptance criterion of ≤15%.

In addition, the test item was found to be stable in application formulations when kept 8 days in the refrigerator (5 ± 3 °C) due to recoveries which met the variation limit of 10% from the time-zero (homogeneity) mean value.

In conclusion, the results indicate the accurate preparation and storage of the test item Gel All DX in vehicle during the study.

Duration of treatment / exposure:

46 days for males and approximately 7 weeks for females.

Frequency of treatment:

Daily, at approximately 24 hour intervals.

Details on study schedule:

Acclimatization: 5 days minimum
First Test Item Administration: Day 1 of pre-pairing
Pre-Pairing: 14 days
Pairing: 14 days maximum
Gestation (females): Approximately 21 days
Treatment ends: On day before sacrifice (males) and on day 3 post partum (females)
Necropsy: After 46 days of treatment (males). Females and pups on Day 4 post partum.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control group

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

48 males (12 per group) and 48 females (12 per group)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous 14-Day Dose Range-Finding Study in the Han Wistar Rat, using dose levels of 0, 100, 300 and 1000 mg/kg/day, resulting in a NOAEL of 1000 mg/kg/day.

Positive control:

None.

Parental animals: Observations and examinations:

VIABILITY/MORTALITY:
Twice daily

CLINICAL SIGNS:
Daily cage-side clinical observations (once daily, during acclimatization and up to day of necropsy). Additionally females were observed for signs of difficult or prolonged parturition, and behavioral abnormalities in nesting and nursing.

FOOD CONSUMPTION:
Males: Weekly during pre-pairing and after pair-ing periods.
Females: Weekly during pre-pairing, gestation period days 0 - 7, 7 - 14 and 14 - 21 and lactation period days 1 - 4.
No food consumption was recorded during the pairing period.

BODY WEIGHTS:
Recorded daily from treatment start to day of necropsy.

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight

Litter observations:

The litters were examined for litter size, live births, still births and any gross anomalies. The sex ratio of the pups was recorded. Pups were weighed individually (without identification) on days 0 (if possible), 1 and 4 post partum.

Postmortem examinations (parental animals):

PATHOLOGY:
Males were sacrificed when they were no longer needed for the assessment of reproductive effects (after 46 days of treatment). Dams were sacrificed on day 4 post partum.

If birth did not occur, the dam was sacrificed on day 25 post coitum.

NECROPSY:
At the scheduled sacrifice, all animals were weighed and sacrificed by an injection of sodium pentobarbital. All P generation animals were exsanguinated.

All parent animals, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.

For the parent animals, special attention was directed at the organs of the reproductive system.

Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

The number of implantation sites and corpora lutea was recorded for all dams with litters. The uteri of non-pregnant females were placed in a solution of ammonium sulfide to visualize possible hemorrhagic areas of implantation sites

ORGAN WEIGHTS:
At the scheduled sacrifice, organs were weighted and preserved as listed below. Organs were trimmed from any adherent tissue, and their wet weight taken.
- Epididymides (fixed in modified Davidson's solution)
- Testes (fixed in modified Davidson's solution)

HISTOTECHNIQUE:
Initially, all organ and tissue samples listed below from control and high dose groups, any decedents and any animals failing to produce a pregnancy were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with hematoxylin and eosin. Additionally, the testis and epididymides were stained by PAS. Special stains were used at the discretion of the study pathologist.

- Epididymides (fixed in modified Davidson's solution)
- Ovaries
- Uterus (incl. oviducts, cervix and vagina)
- Prostate gland and seminal vesicles incl. coagulating glands
- Testes (fixed in modified Davidson's solution)
- All gross lesions

HISTOPATHOLOGY:
Slides of all organs and tissues collected at terminal sacrifice from the animals of the control and high-dose groups were examined by the pathologist. The same applied to all occurring gross lesions and to all animals, which died spontaneously, or had to be terminated in extremis.

Qualitative assessment of the male reproductive organs was performed. Special emphasis was made on the stages of spermatogenesis and histopathology of interstitial cell structure.

A histopathology peer review was performed.

Postmortem examinations (offspring):

Pups were sacrified on day 4 post partum.

All pups, except those excessively cannibalized, were examined macroscopically for any structural changes, either at the scheduled necropsy or during the study if death occurred to establish, if possible, the cause of death.

Statistics:

The following statistical methods were used to analyze food consumption, body weights and reproduction data:

• Means and standard deviations of various data were calculated.

• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.

• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.

• Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

From the on-line recorded reproduction data, the following parameters were calculated: fertility indices, mean precoital time, post-implantation loss.
For reproduction data, group mean values were calculated both on a litter basis and on a percentage per group basis.

Offspring viability indices:

From the on-line recorded reproduction data, the following parameters were calculated: mean litter size, pup sex ratios and viability indices.
Mean pup weights were calculated from the individual weights both on a per group and on a per litter basis.

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

VIABILITY/MORTALITY:
No test item related deaths were noted during the study.

One male given 1000 mg/kg body weight/day (no. 48) was found dead on day 12 of the after pairing period. Prior to its death a decrease in food consumption and body weight loss were noted for several days however no clinical signs or test item-related macroscopical findings were noted, which would clarify the cause of this death. Because of an isolated occurrence, it was considered to be unlikely related to toxicity of the test item.

CLINICAL SIGNS OR OBSERVATIONS:
There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to administration of the test item.

Incidentally, hair loss and scabs were observed in one male (no. 24) at the dose level of 100 mg/kg bw/day and in one female (no. 84) at the dose level of 300 mg/kg bw/day. Hair loss was also noted in one female (no. 96) at the dose level of 1000 mg/kg bw/day.

Further, one female (no. 74) at the dose level of 300 mg/kg bw/day was observed with ruffled fur and one further female (no. 93) at the dose level of 1000 mg/kg bw/day was observed with ruffled fur and decreased activity on day 22 post coitum. Female no. 93 had ruffled fur also during lactation and it neglected its litter.

For both females, the onset of the clinical sign corresponded with the day of parturition. Therefore, as these findings were isolated incidents and were distributed across the dose levels, it was considered that they were attributable to parturition and not to administration of the test item.

FOOD CONSUMPTION OF MALES:
Pre-pairing and Afer Pairing Periods:
There was no effect of the test item on the group mean amount of food consumed by males at any dose level over the course of this study, when compared to control animals.

Mean food consumption at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 23.3, 23.4, 22.8 and 24.2 g/animal/day during pre-pairing and 19.1, 20.2, 19.2 and 20.2 g/animal/day during after pairing periods.

At the dose level of 1000 mg/kg bw/day, statistically significantly higher food consumption was noted from day 15 to 21 of after pairing period. Mean food consumption during this period was 20.8 g/animal/day at this dose level compared to 17.9 g/animal/day in the control group. A slight decrease of food consumption in the control group contributed to this effect and therefore it was considered not to be test item-related but a result of biological variability.

FOOD CONSUMPTION OF FEMALES:
Pre-pairing, Gestation and Lactation Periods:
There was no effect of the test item on the group mean amount of food consumed by females at any dose level over the course of this study, when compared to control animals.

Mean food consumption at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 15.6, 15.2, 15.4 and 15.9 g/animal/day during pre-pairing, 20.8, 19.9, 20.3 and 21.2 g/animal/day during gestation and 26.4, 23.1, 25.0 and 23.5 g/animal/day during lactation periods.

BODY WEIGHTS OF MALES:
Pre-Pairing, Pairing and After Pairing Periods:
Group mean body weight and body weight gain for males, were considered to be unaffected by administration of the test item over the duration of the study, when compared to control animals.

Mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 12%, 14%, 13% and 14% during pre-pairing, 4%, 6%, 6% and 7% during pairing and 6%, 7%, 5% and 6% during after pairing periods.

Statistically significantly higher body weight gain was occasionally observed in all treated groups during the pairing period. The changes were not clearly dose dependent and therefore this observation was considered not to be related to the treatment with the test item but probably be due to incidentally lower body weight gain in the control group.

Statistically significantly lower body weight gain was noted at the dose level of 1000 mg/kg bw/day from day 11 to 14 of after pairing period. The differences to the respective values in the control group were only minor; 2% were noted at the high-dose level and 4% in the control group. During the same period, absolute body weights at the high-dose level were slightly higher than body weights in the control group and therefore this difference was considered to be a result of biological variability and not test item-related.

BODY WEIGHTS OF FEMALES:
Pre-Pairing, Pairing, Gestation and Lactation Periods:
Group mean body weight and body weight gain for females, were considered to be unaffected by administration of the test item over the duration of the study, when compared to control animals.

Mean body weight gain at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day was respectively: 8%, 9%, 9%, 8% during pre-pairing, 57%, 58%, 61% and 60% during gestation and 3%, 3%, 2%, 4% during lactation periods.

MATING PERFORMANCE AND FERTILITY:
No effect on mating performance or fertility was observed at any dose level.

With exception for female no. 84 at the dose level of 300 mg/kg body weight/day and female no. 86 at the dose level of 1000 mg/kg body weight/day, all females mated during the first six days of the pairing period. The precoital time was not affected by the treatment with the test item. Mean (median) precoital times calculated for the first pairing period were 3.3 (3), 3.2 (3), 2.6 (3) and 2.9 (3) days in order of ascending dose levels.

Mating of female nos. 84 and 86 was not observed during the first pairing period. Significant body weight gain was recorded at the end of this period in both females. It was considered that the body weight gain indicated pregnancy after the mating was overlooked. For this reason, females 84 and 86 were not mated with a second male partner. During necropsy it was verified that these females were not pregnant.

Two females in the control group were not pregnant (nos. 50 and 59). Fertility indexes (number of females achieving pregnancy as a percentage of females paired) and conception rates (number of females achieving pregnancy as a percentage of females mated) were 83.3% in the control group and 100% in the remaining groups.

No birth was recorded for one female at the dose level of 1000 mg/kg bw/day (no. 95). At termination on day 25 post coitum, only resorptions/implantation sites were found in this female, proving that she had successfully mated. All remaining females gave birth to living pups. Gestation index (number of females with living pups as a percentage of females pregnant) was 100% in the control group and at the dose levels of 100 and 300 mg/kg bw/day and 90.9% at the dose level of 1000 mg/kg bw/day.

DURATION OF GESTATION:
No effects on duration of gestation were observed at any dose level. Mean duration of gestation was 21.6, 21.3, 21.5 and 21.4 days, for groups given 0, 100, 300 or 1000 mg/kg bw/day, respectively.

CORPORA LUTEA COUNT:
No effects on corpora lutea count were observed at any dose level. Mean number of corpora lutea per dam was 16.4, 15.8, 16.6 and 16.8 in order of ascending dose levels.

IMPLANTATION RATE AND POST-IMPLANTATION LOSS:
At the dose levels of 100 and 300 or 1000 mg/kg bw/day, no effects on implantation rate or post-implantation loss were noted. The overall number of implantations per dam was 12.5, 13.2, 13.7 and 13.4 with a corresponding mean number of post-implantation losses per dam of 1.3, 1.3, 1.5 and 1.0 at the dose level of 0, 100, 300 and 1000 mg/kg bw/day, respectively.


PATHOLOGY (PARENT ANIMALS):
ORGAN WEIGHTS:
No changes in absolute testes and epididymides weights or weights of these organs relative to body weight at any dose level.

MACROSCOPIC FINDINGS:
No test item-related observations were recorded during necropsy at any dose level.

In males, alopecia and eschar were noted in one male (no. 24) in the low-dose group, and advance autolysis was recorded for one male which was found dead (no. 48) at the high-dose level.
In females, shortened left horn was noted in one female (no. 67) at the low-dose level, hardened inguinal mammary gland was noted in one female (no. 82) in the mid-dose group and fetal resorptions were found in one female (no. 95) at the high-dose level.

All these findings were typical of this strain and age of rat and were considered to be incidental.

MICROSCOPIC FINDINGS:
No test item-related observations were recorded in this study.

All findings observed in this study were considered to be of spontaneous background encountered in this strain and type of study.

Dose descriptor:

NOEL

Remarks:

reproduction/developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect on mating performance or fertility was observed at any dose level.

Dose descriptor:

NOEL

Remarks:

general toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects on any parameter measured, at any dose level.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

OBSERVATIONS (LITTER DATA - FI PUPS):

LITTER SIZE AT FIRST LITTER CHECK:
No effects on litter size were observed at any dose level. Mean number of pups per dam was 11.2, 11.9, 12.3 and 12.4 in order of ascending dose levels. Birth index (number of pups born alive as a percentage of implantations) was 89.6%, 90.5%, 89.4% and 92.5% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively.

POSTNATAL LOSS DAYS 0 - 4 POST PARTUM:
At the dose level of 1000 mg/kg body weight/day, statistically significantly increased post natal loss was recorded; 13 pups at this dose level were missing or found dead on day 2 or 3 of the lactation period whereas no postnatal loss was recorded in the control group. Viability index (number of pups alive on day 4 post partum as a percentage of pups born alive) was 100.0%, 97.2%, 99.3% and 89.5% at the dose levels of 0, 100, 300 and 1000 mg/kg bw/day, respectively. Reduction of viability index at the high-dose level was statistically significant.

All pups lost during the lactation at the high dose level were confined to the litter of one female (no. 93). As no pup deaths were recorded in any other litter at this dose level the loss of litter no. 93 was considered to be incidental and not related to treatment with the test item.


EXTERNAL EXAMINATION AT FIRST LITTER CHECK AND DURING LACTATION:
No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

During lactation, scab was noted for two pups from two litters (nos. 69 and 71) at the dose level of 100 mg/kg bw/day. In the neglected litter no. 93 at the dose level 1000 mg/kg bw/day, two pups were found with weakened condition and cold to touch on day 2 post partum. These pups were missing on day 3 post partum.

SEX RATIOS:
Pups sex ratio was not affected by exposure to the test item at any dose level.

At first litter check, percentages of male pups were 50%, 57%, 44% and 48%, in order of ascending dose level.

BODY WEIGHTS TO DAY 4 POST PARTUM:
No effects on pup body weights or body weight gain were noted at any dose level.

Mean body weights of pups per group on day 1 post partum was 6.43, 6.11, 6.22 and 6.07 g whereas mean body weight gain during lactation was 47.70%, 50.36%, 48.25% and 47.31%, both in order of ascending dose levels

MACROSCOPIC FINDINGS:
No test item-related findings were noted in pups at necropsy at any dose level.

Scab recorded for pup in litter no. 92 at the dose level of 100 mg/kg bw/day during lactation was confirmed during necropsy as sore. No further findings were recorded at any dose level.

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on F1 pups

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reproduction and Breeding Data: Summary of Performance

P Animals Breeding for F1 Litters

 

Group (mg/kg/day)	1 (0)	2 (100)	3 (300)	4 (1000)
Female numbers	49 - 60	61 - 72	73 - 84	85 - 96
Number of females paired	12	12	12	12
Number of females not mated (A)	0	0	1	1
Number of females mated but not pregnant	2	0	0	0
Numbers of pregnant females, which did not deliver any pups (B)	0	0	0	1
Number of females with living pups at first litter check	10	12	11	10
Number of females, which lost their litters (C)	0	0	0	1
Number of females which reared their pups until day 4post partum	10	12	11	9

(A)   Females 84 and 86 were considered to have mated.

(B)   Female 95 had fetal resorptions only.

(C)   Female No. 93 had no surviving pups by day 3 of lactation

Conclusions:

The NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

General

The purpose of the study was to generate preliminary information concerning the effects of Gel All DX on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

 

The test item was administered once daily orally (by gavage) throughout pre-pairing, pairing and after pairing periods for a total of 46 days in males and pre-pairing, pairing, gestation and lactation periods for at least 40 days in females, up to the day before scheduled necropsy.

 

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (olive oil).

Parent Animals

Mortality/Viability

No test item related deaths were noted during the study.

 

One male given 1000 mg/kg body weight/day was found dead on day 12 of the after pairing period. The cause of this death was unclear and, due to an isolated occurrence, unlikely to be related to toxicity of the test item.

Clinical Signs

There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to the administration of the test item.

Food Consumption

There was no effect of the test test item on the group mean amount of food consumed by males or females at any dose level over the course of this study, when compared to control animals.

Body Weights

Group mean body weight and body weight gain for males and females were considered to be unaffected by administration of the test item over the duration of the study.

Reproduction and Breeding Data

Fertility, time course of mating, duration of gestation, corpora lutea count, number of implantation sites, post-implantation loss, liter size and post natal loss were unaffected by administration of the test item at any dose level.

Organs Weights

No changes in absolute testes and epididymides weights or weights of these organs relative to body weight were noted at any dose level.

Macroscopic/Microscopic Findings

No test item-related macroscopical or microscopical findings were recorded at any dose level.

Litter Data - F1 Pups

Findings at First Litter Check and During Lactation

No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

 

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

Pup Weights to Day 4 Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

No test item-related findings were noted in pups at necropsy at any dose level.

Conclusion

Based on these results, NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

13-Jun-2013 to 01-Nov-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance:
IUPAC name: 1,3:2,4-bis-O-((3,4-dimethylphenyl)methylene) D-Glucitol
EC number: 413-110-2
CAS number: 135861-56-2


Target Substance: Bis-O-(benzylidene)-D-glucitol
IUPAC name: 1,3:2,4-bis-O-dibenzylidene-D-glucitol
EC number: 251-136-4
CAS number: 32647-67-9


3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data (and toxicokinetic behaviour assessment) it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Dose descriptor:

NOEL

Remarks:

reproduction/developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effect on mating performance or fertility was observed at any dose level.

Dose descriptor:

NOEL

Remarks:

general toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No test item related effects on any parameter measured, at any dose level.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects on F1 pups

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

Executive summary:

General

The purpose of the study was to generate preliminary information concerning the effects of Gel All DX (structural analogue source substance EC 413-110-2)

on male and female reproductive performance such as gonadal function, mating behavior, conception and parturition.

 

The test item was administered once daily orally (by gavage) throughout pre-pairing, pairing and after pairing periods for a total of 46 days in males and pre-pairing, pairing, gestation and lactation periods for at least 40 days in females, up to the day before scheduled necropsy.

 

The following dose levels were used:

Group 1: 0 mg/kg body weight/day (control group)

Group 2: 100 mg/kg body weight/day

Group 3: 300 mg/kg body weight/day

Group 4: 1000 mg/kg body weight/day

 

A standard dose volume of 5 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (olive oil).

Parent Animals

Mortality/Viability

No test item related deaths were noted during the study.

 

One male given 1000 mg/kg body weight/day was found dead on day 12 of the after pairing period. The cause of this death was unclear and, due to an isolated occurrence, unlikely to be related to toxicity of the test item.

Clinical Signs

There were no clinical signs seen in males or females at any dose level, over the course of the study that were considered to be related to the administration of the test item.

Food Consumption

There was no effect of the test test item on the group mean amount of food consumed by males or females at any dose level over the course of this study, when compared to control animals.

Body Weights

Group mean body weight and body weight gain for males and females were considered to be unaffected by administration of the test item over the duration of the study.

Reproduction and Breeding Data

Fertility, time course of mating, duration of gestation, corpora lutea count, number of implantation sites, post-implantation loss, liter size and post natal loss were unaffected by administration of the test item at any dose level.

Organs Weights

No changes in absolute testes and epididymides weights or weights of these organs relative to body weight were noted at any dose level.

Macroscopic/Microscopic Findings

No test item-related macroscopical or microscopical findings were recorded at any dose level.

Litter Data - F1 Pups

Findings at First Litter Check and During Lactation

No test-item related findings were noted in pups at first litter check or during lactation at any dose level.

 

Pups sex ratio was not affected by exposure to the test item at any dose level.

 

Pup Weights to Day 4 Post Partum

No effects on pup body weights or body weight gain were noted at any dose level.

Macroscopic Findings

No test item-related findings were noted in pups at necropsy at any dose level.

Conclusion

Based on these results, NOEL (No Observed Effect Level) for both general toxicity in males and females for reproduction/ developmental toxicity was considered to be 1000 mg/kg bw/day, the highest dose level tested.

It is proposed that this result can be used in the assessment of the target substance.

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 January 2009 to 7 August 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Wistar Han™:HsdRccHan™:WIST strain rats, obtained from Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: (P) Males: Approximately 6 weeks old; Females: Approximately 10 weeks old.
- Weight at study initiation: (P) Males: 230 to 285 g; Females: 188 to 256 g.
- Housing: The animals were housed in groups of four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): A pelleted diet (Rodent 2018C Teklad Global Certified Diet Harlan Laboratories UK Ltd., Oxon, UK), ad libitum.
- Water (e.g. ad libitum): Mains drinking water, ad libitum.
- Acclimation period: 7 days for males and 15 days for females.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2ºC.
- Humidity (%): 55 ± 15%.
- Air changes (per hr): At least 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting, 12 hrs light/12 hrs dark.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was prepared at the appropriate concentrations as a suspension in Arachis oil BP. The formulations were shown to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at approximately 4ºC in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The prepared formulations in the vehicle were within acceptable limits for the purpose of this study.
- Concentration in vehicle: 16.7 mg/ml, 50 mg/ml and 167 mg/ml.
- Amount of vehicle (if gavage): 6 ml/kg.

Details on mating procedure:

Males were paired with females on a one male: one female basis within each dose group, for a period of up to twenty-one days. Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. The presence of sperm within the vaginal smear and/or vaginal plug in situ was taken as positive evidence of mating and the males were subsequently returned to their original holding cages. Mated females were housed individually during the period of gestation and lactation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Summary; The concentration of 4MDBS in the test material formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

Materials and methods; The test material formulations were diluted with dimethylformamide to give a final, theoretical test material concentration of approximately 0.1 mg/ml.
Standard solutions of test material were prepared in dimethylformamide at a nominal concentration of 0.1 mg/ml.
The standards and samples were analysed by HPLC using the following conditions:
HPLC : Agilent Technologies 1050/1200, incorporating autosampler and workstation
Column : Sonoma 5μ C18 (250 x 4.6 mm id)
Mobile phase : Tetrahydrofuran:0.1% orthophosphoric acid (45:55 v/v)
Flow-rate : 1 ml/min
UV detector wavelength : 220 nm
Injection volume : 25 μl
Retention time : ~ 7.2 mins

The test material formulations were mixed thoroughly and samples were taken from the top, middle and bottom of the container, shaking between sampling. Sampling was performed in triplicate.
The test material formulations were sampled and analysed initially and then after storage at approximately 4ºC in the dark for 14 days. The test material formulations were sampled and analysed within 3 days of preparation.

Results; A range of standard solutions covering the concentration range 0 to 0.1541 mg/ml, were prepared and analysed. The detector response was shown to be linear up to 0.1541 mg/ml.
Analysis of the solvent and a blank Arachis Oil BP (control) produced no signal that interfered with the signal due to the test material.
The analytical method has been considered to be sufficiently accurate for the purpose of this study. The test sample results have not been corrected for recovery.

Conclusion; The analytical method has been satisfactorily validated in terms of linearity, specificity and accuracy for the purposes of the study.

Duration of treatment / exposure:

Repeated oral administration for up to 19 consecutive weeks.

Administration by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats.

On completion of the dosing period all males were killed. Females were killed on Day 21 post partum.

Frequency of treatment:

Daily (except during littering/parturition for females).

Details on study schedule:

- Age at mating of the mated animals in the study: Male: Approximately 16 weeks old; Female: Approximately 12 weeks old.

Sequence of study:
i) Groups of 24 male and 24 female animals were treated at the appropriate dose level throughout the study (except during littering/parturition for
females).
ii) A vaginal smear was prepared daily for twenty-one days prior to pairing for all females.
iii) Following ten weeks of treatment for males and two weeks of treatment for females, all animals were paired on a 1 male: 1 female basis within each dose group for a maximum of twenty-one days.
iv) Following evidence of mating, the males were returned to their original cages and females were transferred to individual cages.
v) Pregnant females were allowed to give birth and maintain their offspring until Day 21 post partum. Evaluation of each litter size, litter weight, mean offspring weights and clinical observations were also performed during this period.
vi) At Day 21 post partum, wherever possible, one male and one female offspring were selected for the evaluation of sexual maturation. At Day 21 post partum, all post partum females and remaining offspring were killed and examined macroscopically. Females which were not pregnant or failed to mate were also terminated.
vii) Following completion of the female gestation and lactation phases, the male dose groups were killed and examined macroscopically. Selected organs were weighed and/or preserved for histopathological examination for males. A sample of epididymal semen was analysed for sperm motility and a sample of semen was prepared for morphological assessment. Testicular and epididymal samples were frozen for subsequent homogenisation resistant spermatid enumeration.

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

control

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Low group

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Intermediate group

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High group

No. of animals per sex per dose:

24 animals per sex per dose (including control group).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen based on previous repeat dose toxicity data.

Positive control:

No.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays.
- Cage side observations checked: Overt signs of toxicity, ill-health or behavioural change.

BODY WEIGHT: Yes
- Time schedule for examinations:
(Male) On Day 1 (prior to treatment) and then weekly until termination.
(Female)On Day 1 of treatment and at weekly intervals during the pre-mating phase. Mated females were weighed on Day 0, 7, 14 and 21 post coitum and on Days 1, 4, 7, 14 and 21 of lactation.

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly for each cage group for both sexes until pairing. Dietary intake for males was recorded weekly following the pairing phase until termination. Following confirmation of mating, dietary intake for females was recorded on Days 0 to 7, 7 to 14 and 14 to 21 post coitum and Days 1 to 4, 4 to 7, 7 to 14 and 14 to 21 of lactation.
- Food consumption for each animal determined and mean daily diet consumption: Yes, ratio of bodyweight change/dietary intake.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily, by visual inspection.

REPRODUCTION:
MATING
Cage tray-liners were checked each morning for the presence of ejected copulation plugs and each female was examined for the presence of a copulation plug in the vagina. A vaginal smear was prepared for each female and the stage of the oestrous cycle or the presence of sperm was recorded. On a confirmation of positive evidence of mating, the males and mated females were housed individually.

PREGNANCY AND PARTURITION
Each pregnant female was observed at approximately 08:30, 12:30 and 16:30 hours and around the period of expected parturition. Observations were carried out at approximately 08:30 and 12:30 hours at weekends and public holidays. The following was recorded for each female:
) Date of pairing/mating
ii) Date and time of observed start of parturition
iii) Date and time of observed completion of parturition
iv) Duration of gestation

Oestrous cyclicity (parental animals):

Prior to pairing of females for the mating phase, a vaginal smear was taken daily for 21 days and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain. The smears were examined microscopically and the stage of oestrous was recorded.

Sperm parameters (parental animals):

Parameters examined in male parental generations: Testes weight, epididymis weight, sperm count in testes, sperm concentration, motility, morphology and spermatid count in epididymides.

Litter observations:

STANDARDISATION OF LITTERS
No.

PARAMETERS EXAMINED
On completion of parturition, the number of live and dead offspring was recorded. On Day 1 post partum, all surviving offspring within each litter were individually identified using ink tattoos on the feet (and tails).
For each litter the following was recorded:
i) Number of offspring born
ii) Number of offspring alive recorded daily.
iii) The sex of individual offspring was recorded on Days 1, 4, 7, 14, 21 post partum.
iv) Clinical condition of offspring from birth to weaning
v) Surface righting and ano-genital distance on Day 1.
vi) Individual offspring and total litter weights on Days 1, 4, 7, 14 and 21 post partum
vii) Necropsy findings of offspring

Selected F1 - Sexual Maturation:
Selected F1 offspring were observed for sexual development. For females the day of appearance of vaginal opening (separation of the labia) was recorded; for males the day of separation of the prepuce from the glans penis was recorded. The bodyweight for each individual at the time of sexual maturation was also recorded.


GROSS EXAMINATION OF DEAD PUPS:
Yes, for a full external and internal examination. Any macroscopic abnormalities were examined.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: On completion of the dosing period all males were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
- Maternal animals: Females were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination on Day 21 post partum.

GROSS NECROPSY / ORGAN WEIGHTS
- Gross necropsy consisted of a full external and internal examination for all animals. Any macroscopic abnormalities were recorded.
In addition, the corpora lutea of all ovaries from pregnant females were counted at necropsy. The uterine implantation sites were counted. In the case of non-pregnant females, the procedure was enhanced by staining the uteri with a 0.5% ammonium polysulphide solution where applicable (Salewski 1964).
(Organ weights) The following organs, removed from adult animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: left cauda epididymis, epididymides, kidneys, liver, ovaries, prostate, pituitary, seminal vesicles (with coagulating gland and fluids), testes and uterus (with cervix and oviducts).

HISTOPATHOLOGY
The following tissues were preserved from all males and females from each dose group, in buffered 10% formalin: coagulating gland, right epididymis (in Bouins fluid and then in 70% IMS after approximately forty-eight hours), ovaries, pituitary gland, prostate, seminal vesicles, right testis, uterus (with oviducts) and cervix, and vagina.
Microscopic examination was conducted by the Study Pathologist.

SEMEM ASSESSMENT:
At necropsy of adult males a semem assessment was performed.

Postmortem examinations (offspring):

SACRIFICE
All surviving offspring were terminated via carbon dioxide asphyxiation.

GROSS NECROPSY
- Gross necropsy consisted of a full external and internal examination. Any macroscopic abnormalities were recorded.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.

Statistics:

Data were processed to give group mean values and standard deviations where appropriate. Organ weight (absolute and relative to terminal bodyweight), weekly bodyweight gain, litter weights and offspring bodyweights were assessed for dose response relationships by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene’s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dennett’s test. Where Levene’s test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney ‘U’ test.
The non-parametric methods were also used to analyse implantation loss, offspring sex ratio and developmental landmarks and reflexological responses.
Probability values (P) are presented as follows:
P < 0.001 ***
P < 0.01 **
P < 0.05 *
p ≥ 0.05 (not significant)
Histopathology data were analysed using the following methods to determine significant differences between control and treatment groups for the individual sexes:
1. Chi-squared analysis for differences in the incidence of lesions occurring with an overall frequency of 1 or greater.
2. Kruskal-Wallis one-way non-parametric analysis of variance for the comparison of severity grades for the more frequently observed graded conditions.
Probability values (P) were calculated as follows:
P < 0.001 +++ --- ***
P < 0.01 ++ -- **
P < 0.05 + - *
P < 0.1 (+) (-) (*)
p ≥ 0.1 N.S. (not significant)
Plus (+) signs indicate positive differences from the control group and minus (-) signs indicate negative differences. Asterisks refer to overall differences between group variation which is non-directional.

Reproductive indices:

Oestrous cycle
The stage of oestrus were classified according to the following criteria:
Dioestrus (D): Predominantly leucocytes present although some epithelial and cornified cells can be seen.
Proestrus (P): Predominantly epithelial cells, usually in significant numbers.
Early Oestrus (E1): Predominantly cornified cells, usually seen as small groups or isolated cells.
Late Oestrus (E2): Predominantly cornified cells usually seen as clumps of cells.
Metoestrus (M): Large numbers of leucocytes with discrete clumps of cornified cells.

The oestrous cycles are classified according to the following criteria:
Normal oestrous: The pattern of daily stages of oestrous show a four to five day cycle, which is generally repeated over 21 days.
Extended oestrous: The observation of a predominance of epithelial/cornified cells for more than two days for more than one oestrous cycle.
Extended dioestrous: The predominant cell type is the leucocyte for more than three consecutive days over more than one oestrous cycle.
Irregular cycle: An irregular length of oestrous cycle is observed over the 21 day evaluation period.
Acyclic - No evidence of an oestrous cycle is observed over the 21 day evaluation period.

Mating performance
i) Pre-coital Interval
Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

ii) Fertility Indices
Mating Index (%) = (Number of animals mated÷Number of animals paired) x 100
Pregnancy Index (%) = (Number of pregnant females÷Number of animals mated) x 100

Offspring viability indices:

Live Birth and Viability Indices
Live Birth Index (%) = (Number of offspring alive on Day 1 ÷ Number of offspring born) x 100
Viability Index 1 (%) = (Number of offspring alive on Day 4 ÷ Number of offspring alive on Day 1) x 100
Viability Index 2 (%) = (Number of offspring alive on Day 7 ÷ Number of offspring alive on Day 4) x 100
Viability Index 3 (%) = (Number of offspring alive on Day 14 ÷ Number of offspring alive on Day 7) x 100
Viability Index 4 (%) = (Number of offspring alive on Day 21 ÷ Number of offspring alive on Day 14) x 100
Viability Index 5 (%) = (Number of offspring alive on Day 21 ÷ Number of offspring alive on Day 1) x 100

Sex Ratio
Sex Ratio (% males) = (Number of male offspring ÷ Total number of offspring) x 100

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition).
There were no further unscheduled deaths during the study.

There were no clinical signs considered to be attributable to systemic toxicity in terminal kill animals. An episode of increased salivation was evident immediately before dosing for one male treated with 100 mg/kg/day on Day 51. Observations of this nature are commonly observed following the oral administration of a slightly unpalatable test material formulation, and in the absence of a similar effect seen at the high dose group the intergroup difference was not considered to represent a systemic effect of treatment. One female from all treatment groups showed instances of pallor of the extremities and one female treated with 100 mg/kg/day also showed hunched posture and pilo-erection. These observations were recorded around the time of parturition and were considered to be due to the littering process rather than a systemic effect of treatment.

The remaining clinical signs evident included generalised fur loss, staining of the external body surface, a damaged tail tip, chromodacryorrhea and scab formation. These were observed throughout the control and/or treatment groups and were considered to be low incidence findings occasionally observed in studies of this type and are unrelated to test material toxicity.

The male treated with 1000 mg/kg/day that was killed in extremis on Day 71 showed loss of righting and blink reflex, hypothermia, pallor of the extremities, hunched posture and lethargy.

BODY WEIGHT (PARENTAL ANIMALS)
There were no treatment-related effects detected in male bodyweight development. No adverse effects on bodyweight change were evident for treated females when compared to controls during the pre-mating, gestation and lactation phases of the study. Statisical analysis did not reveal any significant intergroup differences.

FOOD COMSUMPTION (PARENTAL ANIMALS)
No toxicologically significant effects were observed in males/females.
Males treated with 1000 mg/kg/day showed a statistically significant increase in food intake during Week 3, 6 and 16. An increase in dietary intake is not considered to be of toxicological importance.
Females treated with 1000 mg/kg/day showed a statistically significant increase in food consumption during the second week of gestation. An increase in dietary intake is not considered to be of toxicological importance. No adverse effects on dietary intake were evident for treated females when compared to controls throughout lactation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No adverse effect on oestrous cycle was detected (in most of the cases, lasting 4 or 5 days).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No toxiciologically significant effects were detected.
Males treated with 1000 mg/kg/day showed a statistically significant reduction in progressive motility and testes spermatid count. In the absence of any histology correlates or an effect on fertility the intergroup differences were considered to be of no toxicological importance.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No adverse effects on mating performance were detected for treated animals when compared to controls. Pre-coital intervals for the majority of paired animals were between 1 to 5 days. Two control pairs mated nine and thirteen days following pairing, two pairs from the 100 mg/kg/day dose group mated eight and eighteen days following pairing, two pairs treated with 300 mg/kg/day mated eight and nineteen days following pairing and four pairs treated with 1000 mg/kg/day mated seven, thirteen, sixteen and seventeen days following pairing. Positive evidence of mating was not confirmed for two pairs treated with 300 mg/kg/day, both of which however were pregnant and produced a litter.

There were no adverse effects detected on fertility or pregnancy status for treated animals when compared to controls. There was three non-pregnant female observed in the 100 mg/kg/day dose group, four non-pregnant females observed in the 300 mg/kg/day dose group and one non-pregnant female observed in the 1000 mg/kg/day dose group. Two females treated with 1000 mg/kg/day did show evidence of pregnancy however no offspring were observed to be born.

No treatment-related effects on the lengths of the gestation period were detected for treated females when compared to their concurrent controls. The majority of pregnant females showed gestation lengths between 21½ and 23 days.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects were detected in the organ weights measured.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no toxicologically significant macroscopic abnormalities detected in terminal kill animals.

One male treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day had small testes and epididymides at necropsy. The male treated with 300 mg/kg/day also had a turgid left testis. A further male treated with 300 mg/kg/day and one male treated with 100 mg/kg/day had small testes at necropsy. One female treated with 1000 mg/kg/day had a fluid filled sac encasing the right ovary. In the absence of any histology correlates detected in the testes, epididymides or ovaries the intergroup differences were considered to be of no toxicological significance. One female treated with 100 mg/kg/day had a fluid filled sac encasing the left kidney. A further female from this treatment group also had enlarged implantations on the left horn of the uterus. In the absence of a dose related response or any histology correlates the intergroup differences were considered to be of no toxicological importance. One control female, one female treated with 100 mg/kg/day, one female treated with 1000 mg/kg/day and one male treated with 300 mg/kg/day had a nodule of the median lobe of the liver protruding through the diaphragm. This was considered to be due to physical damage
occurring during the necropsy procedure and not due to systemic toxicity.
A summary incidence of necropsy findings for males is given in Table 1 and for females is given in Table 2 as attached. Individual data are given in tables 3 and 4 as attached.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related microscopic abnormalities were detected.
A summary incidence of histopathological findings is given in Table 5 and Table 6 as attached. Individual data are given in tables 7 and 8 as attached.

OTHER FINDINGS (PARENTAL ANIMALS)
WATER COMSUMPTION
No toxicologically significant effects were observed in males/females.

Dose descriptor:

NOAEL

Remarks:

reproductive and developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically significant reproductive effects in any parameters examined at any dose levels tested.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

LITTER RESPONSES
With the exception of two pairs treated with 300 mg/kg/day, all animals mated. In total, twenty-three control females, nineteen 100 mg/kg/day females, nineteen 300 mg/kg/day females and nineteen females treated with 1000 mg/kg/day gave birth to a live litter and successfully reared young to Day 21 of age. Total litter losses were evident for one control female, one 100 mg/kg/day female, one 300 mg/kg/day and two females treated with 1000 mg/kg/day.

The following assessment of litter response is generally based on those litters reared to termination on Day 21 post partum, although data available for females showing a total litter loss has also been taken into consideration, where considered appropriate.


LITTER SIZE AND VIABILITY (OFFSPRING)
There were no treatment-related differences in the number of corpora lutea, implantation sites, pre-implantation losses or post implantation losses for treated animals when compared to controls. Litter sizes and the viability of the litters from treated animals were comparable to litters delivered by control dams. The ratio of males to females within each dose group was also similar for treated groups when compared to controls. Statistical analysis of the data did not reveal any significant intergroup differences.

CLINICAL SIGNS (OFFSPRING)
No clinically observable signs of toxicity were detected in offspring from the treated groups. The clinical observations observed throughout the control and treated groups were low incidence findings observed in studies of this type and were considered not to be related to treatment with the test material. The five total litter losses detected in a control female, a female treated with 100 mg/kg/day, a female treated with 300 mg/kg/day and a female treated with 1000 mg/kg/day were isolated and are occasionally observed in reproductive studies. Due to the presence of a total litter loss in the control group, these litter losses were considered to be unrelated to treatment.

BODY WEIGHT (OFFSPRING)
There were no adverse effects detected for litter weights or mean offspring bodyweights for offspring from treated animals when compared to offspring from control animals. Statistical analysis of the data did not reveal any significant intergroup differences.

SEXUAL MATURATION (OFFSPRING)
There were no treatment-related changes in the attainment of sexual maturation or ano-genital distance. Statistical analysis of the data did not reveal any significant intergroup differences.

GROSS PATHOLOGY (OFFSPRING)
No treatment-related macroscopic abnormalities were detected for both interim death and terminal kill offspring throughout the control and treated groups. The incidental findings observed throughout the control and treatment groups were occasionally observed low incidence findings in animals of the age employed and not considered to represent treatment-related changes in the affected tissues or organs.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A NOAEL of 1000 was established for developmental toxicity in progeny

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

Executive summary:

Introduction.

The study was designed to investigate the effects of the test material when administered throughout the reproductive cycle of the rat to assess prenatal and postnatal development, and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).

Methods.

The test material was administered by gavage to three groups, each of twenty four male and twenty-four female Wistar Han™: HsdRccHan™: WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. Oestrous cycle assessment was performed daily for three weeks prior to mating.

After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum. The ano-genital distance was recorded for all F1 generation offspring on Day 1 post partum.

All males were terminated following the completion of a successful mating. All surviving females and unselected offspring were terminated on Day 21 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Selected offspring from each litter (where applicable) were evaluated for sexual maturation.

Results.

Mortality. One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition). There were no further unscheduled deaths during the study.

Clinical Observations. No clinically observable signs of toxicity were detected in terminal kill animals.

Bodyweight. No adverse effect on bodyweight gain was detected for males throughout the treatment period or for females throughout maturation, gestation or lactation.

Food Consumption. No adverse effect on food consumption was detected.

Water Consumption. No intergroup differences were detected.

Oestrous Cycle Assessment. There were no adverse effects on oestrous cycle assessments.

Mating. No adverse effects on mating performance was observed for treated animals when compared to controls.

Fertility & Pregnancy. There were no adverse effects of treatment on fertility or pregnancy rates between treated and control groups.

Gestation. No treatment-related effects were evident in the length of gestation for treated females when compared to controls.

Litter Responses. There was no adverse effect of treatment of the parent female on implantation rate, in-utero survival, litter size on Day 1 and subsequent offspring survival to weaning at dose levels of 100, 300 or 1000 mg/kg/day. Sex ratio of the offspring was essentially similar in all groups throughout lactation. There was no obvious adverse effect of treatment of the parent female on litter weights or offspring weight on Day 1 or subsequent bodyweight gain to weaning at 100, 300 or 1000 mg/kg/day.

Offspring Ano-genital Distances. No adverse effect was detected on ano-genital distance between treated and control groups.

Necropsy. No toxicologically significant effects were detected in terminal kill animals.

Organ Weights. No treatment-related effects were detected in the organ weights measured.

Sperm Analysis. No toxicologically significant effects were detected in the sperm parameters measured.

Histopathology. No treatment-related microscopic abnormalities were detected.

Conclusion. The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of

the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive

toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

Reference 4

Endpoint:

one-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:

Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
It is proposed that the structural similarity and properties of the target substance and the structural analogue (sources substance) are sufficiently close for there to be a reasonable expectation of similar effects.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance:
IUPAC name: 1-(2,6-bis(4-tolyl)-1,3-dioxano(5,4-d)-1,3-dioxan-4-yl)ethane-1,2-diol
EC number: 402-950-5
CAS number: 87826-41-3

Target Substance: Bis-O-(benzylidene)-D-glucitol
IUPAC name: 1,3:2,4-bis-O-dibenzylidene-D-glucitol
EC number: 251-136-4
CAS number: 32647-67-9


3. ANALOGUE APPROACH JUSTIFICATION
Based on the structural similarity of the source substances and target substance, similarity of physic-chemical properties and similarity in experimental (eco)toxicological test data (and toxicokinetic behaviour assessment) it is concluded that target substance and the structural analogue (source substance) are sufficiently close for there to be a reasonable expectation of similar effects, for the endpoints where results have been read-across.

4. DATA MATRIX
Please see Section 13 for "Read-across justification to support the REACH registration of Bis-O-(benzylidene)-D-glucitol (EC 251-136-4) at 10-100 tpa" for full details.

Reason / purpose for cross-reference:

read-across source

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Dose descriptor:

NOAEL

Remarks:

reproductive and developmental toxicity

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicologically significant reproductive effects in any parameters examined at any dose levels tested.

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: A NOAEL of 1000 was established for developmental toxicity in progeny

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

Executive summary:

Introduction.

The study was designed to investigate the effects of the test material structural analogue source substance EC 402 -950 -5)

when administered throughout the reproductive cycle of the rat to assess prenatal and postnatal development, and complies with the OECD Guidelines for Testing of Chemicals No 415 "One Generation Reproduction Toxicity Study" (Adopted 26 May 1983).

Methods.

The test material was administered by gavage to three groups, each of twenty four male and twenty-four female Wistar Han™: HsdRccHan™: WIST strain rats, at dose levels of 100, 300 and 1000 mg/kg/day. A control group of twenty-four males and twenty-four females was dosed with vehicle alone (Arachis oil).

Clinical signs, bodyweight development, dietary intake and water consumption were monitored during the study. Oestrous cycle assessment was performed daily for three weeks prior to mating.

After ten weeks of treatment for males and two weeks of treatment for females, pairing of animals within each dose group was undertaken on a one male: one female basis, to produce litters. During the lactation phase, daily clinical observations were performed on all surviving offspring, together with litter size. Litter weights and individual offspring weights were also recorded on specific days post partum. The ano-genital distance was recorded for all F1 generation offspring on Day 1 post partum.

All males were terminated following the completion of a successful mating. All surviving females and unselected offspring were terminated on Day 21 post partum. All animals were subjected to a gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Selected offspring from each litter (where applicable) were evaluated for sexual maturation.

Results.

Mortality. One male treated with 1000 mg/kg/day was killed in extremis on Day 71. One female treated with 100 mg/kg/day was killed in extremis on Day 43 (during expected time of parturition). There were no further unscheduled deaths during the study.

Clinical Observations. No clinically observable signs of toxicity were detected in terminal kill animals.

Bodyweight. No adverse effect on bodyweight gain was detected for males throughout the treatment period or for females throughout maturation, gestation or lactation.

Food Consumption. No adverse effect on food consumption was detected.

Water Consumption. No intergroup differences were detected.

Oestrous Cycle Assessment. There were no adverse effects on oestrous cycle assessments.

Mating. No adverse effects on mating performance was observed for treated animals when compared to controls.

Fertility & Pregnancy. There were no adverse effects of treatment on fertility or pregnancy rates between treated and control groups.

Gestation. No treatment-related effects were evident in the length of gestation for treated females when compared to controls.

Litter Responses. There was no adverse effect of treatment of the parent female on implantation rate, in-utero survival, litter size on Day 1 and subsequent offspring survival to weaning at dose levels of 100, 300 or 1000 mg/kg/day. Sex ratio of the offspring was essentially similar in all groups throughout lactation. There was no obvious adverse effect of treatment of the parent female on litter weights or offspring weight on Day 1 or subsequent bodyweight gain to weaning at 100, 300 or 1000 mg/kg/day.

Offspring Ano-genital Distances. No adverse effect was detected on ano-genital distance between treated and control groups.

Necropsy. No toxicologically significant effects were detected in terminal kill animals.

Organ Weights. No treatment-related effects were detected in the organ weights measured.

Sperm Analysis. No toxicologically significant effects were detected in the sperm parameters measured.

Histopathology. No treatment-related microscopic abnormalities were detected.

Conclusion. The administration of 4MDBS by oral gavage to rats for either a period of up to one hundred and thirty three consecutive days for males or at least seventeen consecutive days prior to mating and throughout the gestation and lactation phases of

the reproductive cycle for female rats, did not result in any toxicologically significant reproductive effects at dose levels of 100, 300 or 1000 mg/kg/day. A ‘No Observed Adverse Effect Level’ (NOAEL) of 1000 mg/kg/day was established for reproductive

toxicity including fertility and mating in adults and for developmental toxicity in their subsequent progeny.

It is proposed that this result can be used in the assessment of the target substance.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

It is proposed that the results from the read-across source substances can be used in the assessment of the target substance.

Effects on developmental toxicity

Description of key information

Refer to effects on fertility.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the absence of adverse effects to reproduction/development in OECD 421 and OECD 415 studies conducted on structurally similar substances, the target substance is not classified for reproductive toxicity.