Octanoic acid, monoester with glycerol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

For Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052 -13 -0) a NOAEL for parental fertility of 1000 mg/kg bw/day in rats could be identified.

For Castor oil (CAS No. 8001-79-4) a NOAEL for parental fertility of 5000 mg/kg bw/day in rats and 15000 mg/kg bw/day in mice could be identified.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study, tested with the source substance Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS 91052-13-0). In accordance to the ECHA guidance document ¿Practical guide 6: How to report read-across and categories (March 2010)¿, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance. Read-across from Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052-13-0) for systemic mammalian toxicity endpoints was judged to be justified for the following reasons: This read across substance is also a glyceride, containing mainly C12-14 fatty acid and acetate moiety as well as glycerol. It¿s an organic liquid with a pour point of -8 °C and a melting point of 357.85 °C and a vapour pressure < 0.01 Pa at room temperature. In contrast to the glycerides of the fatty acid glyceride category, it has a higher water solubility of 8.75 mg/L, which might influence its environmental distribution, but not the mammalian metabolism upon systemic uptake. Therefore it is expected to feed into the same mammalian physiological pathways as the members of the fatty acid glyceride category, like citric acid cycle, sugar synthesis and lipid synthesis.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Principles of method if other than guideline:

After acclimatisation, four Main groups of 10 male and 5 female Wistar Han rats were exposed by oral gavage to the test substance at 0, 100, 300 and 1000 mg/kg/day. An additional 5 Recovery group males and females in the control and high dose group were allowed 14 days of recovery.
An additional 10 females were added to each group for the assessment of reproduction and developmental toxicity. Main and Recovery animals were exposed for at least 28 days from start of treatment up to termination or start of recovery. Females used for the assessment of reproduction/developmental toxicity were exposed for 41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France.
- Age at study initiation: Approximately 12 weeks.
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days prior to start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range: 19.7 - 21.9°C)
- Humidity (%): 40 - 70% (actual range: 22 - 71%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. NOTOX BV has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at NOTOX.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).

Following a minimum of 14 days of exposure for the males and females, one Repro female was cohabitated with one Main male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was confirmed by evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or or by the appearance of an intravaginal copulatory plug. Detection of mating was not confirmed for animal no. 98 which did deliver. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated
Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating. After 14 days of mating, females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ¿ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analyzed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ¿ 10%) and formulations at the entire range were stable when stored at room temperature for at least 6 hours.

Duration of treatment / exposure:

41-49 days,
i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

once daily for 7 days per week

Details on study schedule:

Offspring were euthanized at the age of 4 days.

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 10-Day dose range finding study

Positive control:

none

Parental animals: Observations and examinations:

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see 7.5.1

Oestrous cyclicity (parental animals):

Uterus epithelium was analyzed histologically for estrus, proestrus and cystic endometrial glands.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: testis weight, epididymis weight, histology of testes
Of the first 5 Main males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis.
The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

Litter observations:

On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals which delivered: on lactation day 5
- Maternal animals which failed to deliver (4 animals): Post-coitum Day 26 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of: see details under 7.5.1
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
see details under 7.5.1

Postmortem examinations (offspring):

Pups surviving to planned termination were killed by decapitation on lactation Day 5.
All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.

Statistics:

see 7.5.1

Reproductive indices:

For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Offspring viability indices:

For each group the following calculations were performed:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive 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

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

as found by histological examination of uterus

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

as found by histological examination of testes

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, as explored by histological examination of testes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Histological examination of the uterus epithelium and endometrial glands did not reveal any treatment related influences on estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment related effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

For further details see 7.5.1

Dose descriptor:

NOAEL

Remarks:

parental fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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:

no effects observed

Number of pups: 432
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

other:

Remarks on result:

other: No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Reproductive effects observed:

not specified

Reference 2

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 352 - 426 g (males; mean: 372 g), 192 - 249 g (females; mean: 224 g)
- Housing: individual in stainless steel cages
- Diet: ad libtum
- Water: ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.1 - 23.2
- Humidity (%): 48 - 61
- Air changes (per hr): more than 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was diluted in appropritate amounts of corn oil for each dose level. Aliquots of the dosing solution corresponding to the amount of daily administration were stored in the dark at 2 - 6 °C. The stability of the dosing solution was 7 days in a refrigerator and 1 day at room temperature. Therefore, the dosing solution was used within 7 days.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance showed low solubility in water.
- Amount of vehicle (if gavage): 5 mL/kg
- Lot/batch no. (if required): V4N3566

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually in a polycarbonate cage with animal bedding for nesting

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

No details reported.

Duration of treatment / exposure:

males: 42 days (14 days prior to mating and 28 days thereafter)
females: 42-52 days (from 14 days before mating to day 4 of lactation)
satellite males and females: 42 days and 14 days post-exposure observation period

Frequency of treatment:

once daily, 7 days/week

Details on study schedule:

- Age at mating of the mated animals in the study: 12 weeks

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

12 females in control and test groups
7 males in control and 1000 mg/kg bw groups
12 males in 100 and 300 mg/kg bw groups
5 animals per sex in satellite control and test groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Two dose range finding studies were performed. 2000 mg/kg bw of test substance was administered for 3 days in male and female rats. No abnormalities were found in general condition and body weight. The second study was performed at dose levels of 0, 330, 100, 300 and 1000 mg/kg bw/day for 14 days. No abnormalities of general condition, body weight, food consumption, hematological findings, blood biochemical findings, gross pathology and organ weight were found. Therefore, 1000 mg/kg bw/day was selected as the highest dose.
- Post-exposure recovery period in satellite groups: 14 days

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first exposure and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Day 1 (before administration), 7, 14, 21, 28, 35 and 42 (before sacrifice)
Females: Day 1 (before administration), 7, 14, during pregnancy on Day 0, 7 14 and 21, during lactation on Days 0 and 4 (before sacrifice)
Satellite males and females: Day 1 (before administration), 7, 14, 21, 28, 35, 42, 49 (Day 7 of recovery period) and 56 (Day 14 of recovery period, before sacrifice)

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Oestrous cyclicity (parental animals):

Numbers of times in estrous before and during administration till mating confirmed.

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight and abnormality, epididymis weight and abnormality, prostate weight and abnormality

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, body weight, physical abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals one day after the last administration (Day 43)
- Maternal animals: All surviving animals on Day 5 of lactation

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed, respectively:
brain, pituitary, thyroid, thymus, lung trachea (after liquid immersion fixation), stomach, intestines, heart, liver, spleen, kidney, adrenal gland, bladder, testis, epididymis, prostate, seminal vesicles, ovaries, uterus, spinal cord (cervical, thoracic, lumbar), sciatic nerve, bone marrow (femur), lymph nodes (cervical lymph node, mesenteric lymph nodes), mammary gland, and other gross abnormalities.
Spermatogenic cycle (Stage II, III, V, VII, and XII) was also investigated.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic).

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the thoracic, and abdominal viscera.

Statistics:

ANOVA, Barlettm Kruskal-Willis, Dunnett, F test, Studen t-test, Aspin-Welch t-test, Mann-Whitney U-test, Fisher's exact test

Reproductive indices:

Copulation index = (No. of pairs with successful copulation/No. of pairs mated) x 100
Fertility index = (No. of pregnant females/No. of pairs with successful copulation) x 100
Gestation index = (No. of females with live pups / No. of pregnant females) x 100

Offspring viability indices:

Delivery index = (No. of pups born / No. of implantation sites) x 100
Live birth index = (No. of live pups on day 0 / No. of pups born) x 100
Viability index = (No. of live pups on day 4 / No. of live pups on day 0) x 100
Sex ratio = total No. of male pups / total No. of female pups

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, males): significant increase was observed (non adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day (satellite group, males): significant increase was observed (non adverse)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

control and 1000 mg/kg bw/day: low incidence of commonly found microscopic changes, evenly distributed between groups (not treatment-related);300 mg/kg bw/day: benign fibroadenoma of the mammary gland in one female (not treatment-related)

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)
No abnormalities and no mortality were observed.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No change of body weight and weight gain was observed during the administration period. During the recovery period, a significant increase in body weight was noted in males at 1000 mg/kg bw/day. This was caused by a tendency of the control group animals to lose weight. One male in control group showed a significant decrease in body weight during the recovery period. However, no other abnormalities were observed in this male.
No change was observed during the administration period in the test groups. A significant increase in food consumption was found in satellite females of the 1000 mg/kg bw/day group on Day 14 of administration. However, it was regarded as an incidental finding since the food consumption of the corresponding control group was relatively small on that day. Therefore, this change was not regarded as a compound-related effect.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No differences were observed between control group and test groups.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Decreased sperm was observed in one male in control group and whose pair was not pregnant.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No changes were observed both in control and test groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
After the administration period, a significant decrease in the absolute weight of seminal vesicles in males given and a significant decrease in relative weight of spleen in females were observed in the 100 mg/kg bw/day group. However, this change was not compound-related because no abnormalities were found at histopathological examination and there was no dose-dependency.
After the recovery period, a significant decrease in relative weight of pituitary in males of the 1000 mg/kg bw/day group and relative weight of thyroid in females of the 1000 mg/kg group bw/day was observed. Nevertheless, this effect was not compound-related because no abnormality was reported regarding these organs after administration period.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No test substance-related changes were found. No abnormalities were found in breeding pairs which were not successful at mating and in those which were successful at mating but not pregnant.
After administration period, reddish thymus was noted in one male of the control group and subcutis mass was found in one female of 300 mg/kg by/day group (see also HISTOPATHOLOGY: NEOPLASTIC). After the recovery period, larger spleen and capsular thickening in spleen was found in one male and reddish area in thymus was observed in one female at 1000 mg/kg bw/day.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No test substance-related changes were observed. There were also no changes regarding spermatogenic cycle.
Myocardial degeneration/fibrosis, foam cell accumulation in lung, mineralization in artery in lung, fatty degeneration of hepatocyte, microgranuloma in liver, solitary cyst in kidney, hyaline cast in kidney, lymphocyte infiltration in cortex of kidney, mineralization of cortico-medullary junction in kidney, fibrosis of cortex in kidney and haemorrhage in thymus were observed both in the control and 1000 mg/kg bw/day groups or only in the control group with low incidence. Hyaline droplet of proximal tubular epithelium in kidney was found in all males of the control and 1000 mg/kg bw/day groups. Brown deposit pigment and extramedullary haematopoiesis in spleen were found in all males and females of the control and 1000 mg/kg bw/day groups. However, there were no differences between control and test group. In the 1000 mg/kg bw/group, lymphocyte interstitium infiltration in prostate was found in one male, interstitial focal inflammation in lung and focal necrosis in liver was observed in one female. These changes occur naturally and were not compound-related.
No abnormalities were found in uterus and ovary in the non-pregnant females and the unsuccessful copulation females of the control and 1000 mg/kg bw/day groups, respectively. Degeneration of seminiferous tubules of testis, decrease in sperm and atrophy in prostate was observed in one control male.
Mass on abdomen was found in a female after 40 days of administration in the 300 mg/kg bw/day group. However, this subcutaneous tumour of the mammary gland was a benign fibroadenoma and generated naturally.

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

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:

effects observed, treatment-related

Description (incidence and severity):

control and test groups: low incidence of commonly found visceral variations (not compound-related)

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
No changes were observed.

CLINICAL SIGNS (OFFSPRING)
No effects were observed.

GROSS PATHOLOGY (OFFSPRING)
Thymic remnant in neck was observed in one, one and two pups in the control, 300 and 100 mg/kg bw/day groups, respectively. Persistent left umbilical artery was found in one and two pups at 100 and 1000 mg/kg bw/day, respectively. Convoluted ureter was found in one, three and two pups at 100, 300 and 1000 mg/kg bw/day, respectively. Dilatation of renal pelvis was observed in one pup at 300 mg/kg bw/day. Dilatation of ureter was observed in one and two pups in 100 and 300 mg/kg bw/day, respectively. However, these changes were regarded as not-compound related, since they could occur naturally and there was no significant difference between control and test groups.

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

not specified

Reference 3

Endpoint:

two-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

09 Jan 2009 - 21 Aug 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance. Justification for grouping of substances and read-across: The study is selected as a suitable study for the assessment of potential reproduction toxicity effects within the Glycerides category. Read-across from Glycerides, castor-oil-mono, hydrogenated, acetates (main component: 12-acetoxy-octadecanoic acid (2,3-diacetoxy)propyl ester [CAS 330198-91-9]) (CAS No. 736150-63-3) is conducted on the basis of Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 by application of the group/category concept (i.e. the physicochemical, toxicological and ecotoxicological properties of a group of substances are likely to be similar or follow a regular pattern as a result of structural similarity). Human health effects with regard to toxicity to reproduction were predicted from data for reference substances within the category by interpolation. The similarities between the members of the Glycerides category are based on the common functional groups, the common precursors and the likelihood of common breakdown products, and a constant pattern in the changing of the potency of the properties across the category. The selected study fulfils the requirements laid down in Annex XI, Item 1.5, of Regulation (EC) No 1907/2006 for read-across, i.e. the results are adequate for the purpose of classification and labelling and/or risk assessment; have adequate and reliable coverage of the key parameters addressed in the corresponding test method referred to in Article 13(3); cover an exposure duration comparable to or longer than the corresponding test method referred to in Article 13(3); and adequate and reliable documentation of the applied method is provided. A detailed justification for the grouping of chemicals and read-across is provided in the technical dossier (see IUCLID Section 13).

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 426 (Developmental Neurotoxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3800 (Reproduction and Fertility Effects)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF, Testing Guidelines for Toxicity Studies (2-1-17), 12 Nohsan No 8147, 2000-11-24

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

THE DEPARTMENT OF HEALTH OF THE GOVERNMENT OF THE UNITED KINGDOM, UK

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD® (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, UK
- Age at study initiation: (P) 7-8 weeks
- Weight at study initiation: (P) 216-361 g (males) and 149-249 g (females)
- Housing: All P animals were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, the animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper. Mated females were housed individually (or with their litter), in polypropylene cages with solid floors and stainless steel lids, furnished with softwood flakes (Datesand Ltd., Cheshire, UK). For the offspring selected to form the F1 generation, this housing procedure was repeated. The P and F1 offspring selected for assessment of developmental neurotoxicity were housed in groups of up to 4 in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: ground diet (Rodent PMI 5002 Diet, BCM IPS Limited, London, UK), ad libitum
- Water: mains drinking water, ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): air-conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Each concentration of the test material was prepared by weighing an appropriate amount and mixing it with a small amount of the required volume of diet. Once this was adequately mixed the formulation was then transferred to a Hobart H800 mixer and mixed with the remaining required volume of diet.

DIET PREPARATION
- Mixing appropriate amounts with: laboratory diet

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 14 days
- Proof of pregnancy: 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.
- After 14 days of unsuccessful pairing replacement of first male by another male or extension of the mating phase to a 3 week.
- After successful mating each pregnant female was caged: individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test material in the dietary admixtures was determined by gas chromatography using an external standard technique. The dietary admixtures were sampled and analysed within 2 weeks of preparation. Homogeneity and stability determinations were performed under Harlan Laboratories Ltd. project number 2384/0006. The results showed that achieved concentrations were between 95 and 121% of nominal concentration.

Duration of treatment / exposure:

(P) Males: 10 weeks during maturation and throughout mating, gestation and until completion of the P female lactation phase.
(P) Females: 10 weeks during maturation and throughout mating, gestation and until Day 21 lactation phases.
(F1) Females: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F1) Males: minimum of 10 weeks during maturation and subsequently throughout mating, gestation and until completion of the F1 female lactation phases. Between weaning and the formal start of the F1 generation, the animals continued to receive their appropriate treated diet.
(F2) Males/ Females: from weaning (Day 21 of age) to termination at Day 70 of age.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 10 weeks
- Due to number of offspring required for developmental neurotoxicity, the study was split into two equal parts. Each group was divided into two equal sub-groups; with animals allocated to the second part being given treated diets eight days later. Chronically, all procedures occurred approximately one week later for the second sub-group compared to the first group.

Remarks:

Doses / Concentrations:
1500, 6000 and 25000 ppm
Basis:
other: nominal in diet: animals in the high dose group initially received 15000 ppm rising to 20000 ppm to 25000 ppm during the maturation phase of each generation

Remarks:

Doses / Concentrations:
82, 324 and 1159 mg/kg bw/day
Basis:
other: mean achieved dose level (P males)

Remarks:

Doses / Concentrations:
146, 587 and 2200 mg/kg bw/day
Basis:
other: mean achieved dose level (P females)

Remarks:

Doses / Concentrations:
109, 435 and 1342 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 males)

Remarks:

Doses / Concentrations:
160, 630 and 2262 mg/kg bw/day
Basis:
other: mean achieved dose level (F1 females)

No. of animals per sex per dose:

28 P males, 28 P females
24 F1 males, 24 F1 females

Control animals:

other: yes, laboratory diet treated with Arachis oil to ensure comparable calorific intake

Details on study design:

- Dose selection rationale: The dietary levels were based on known toxicology data including an earlier preliminary study in the rat (Project number 2384/0006).
- Other: In each generation, animals receiving the high dose level initially received the test material at a concentration of 15000 ppm and this was increased to 20000 ppm and finally 25000 ppm as the study progressed. Animals were receiving the highest inclusion level by the end of the maturation/pre-pairing phase in both generations and therefore were receiving the higher dietary inclusion by the time of the main reproductive assessment.

Positive control:

The study incorporated a positive control for endocrine disruption, DEHP (bis(2-thylhexyl) phthalate), throughout the P generation and until sexual maturation of the F1 ofspring. Animals received an intended dietary inclusion level of 10000 ppm.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily during the week and once daily on weekends and public holidays

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Day 1, prior to treatment, weekly throughout the maturation phase of each generation and continuing for males until termination. Following pairing P/F1 females were weighted daily until mating was evident. Mated females were weighted on Day 0, 7, 14 and 21 post coitum and for females that littered on Day 1, 4, 7, 14 and 21 post partum. P/F1 offspring (selected for post-weaning assessment of developmental neurotoxicity) were weighted on Day 28, 35, 42, 49, 56, 63 and 70 of age. Body weight was recorded for all animals on the Day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE :
- Food consumption was assessed for each cage of adults during the maturation period and for males after mating/pairing. Females showing evidence of mating, food consumption was recorded for the periods Day 0-7, 7-14 and 14-21 post coitum. For females that littered, food consumption was recorded for the period covering Day 1-4, 4-7, 7-14 and 14-21 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: daily by visual inspection for any overt change

Oestrous cyclicity (parental animals):

Prior to pairing of females for the P and F1 mating phases, a vaginal smear was taken daily for twenty-one days and a sample was placed on a glass slide. The smears were allowed to dry and then stained using a diluted giemsa stain and examined microscopically.

Sperm parameters (parental animals):

Parameters examined in P/F1 male parental generations in control and high-dose males: Yes. Testis weight, epididymis weight, spermatid enumeration, sperm motility, sperm morphology.

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in P/ F1 offspring: number and sex of pups, live births, postnatal mortality reported on Day 1, 4, 7, 14 and 21, clinical condition, individual bodyweight, necropsy findings, ano-genital distance and number of visible nipples on Day 11 and 15 and neurobehavioural effects.

GROSS EXAMINATION OF DEAD PUPS:
Yes, dead offspring was subjected to a necropsy examination.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving male F0/F1 animals were killed following completion of the F0 female lactation phase.
- Maternal animals: All surviving F0/F1 females were killed and examined macroscopically at Day 21 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Selected organs (adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions) were weighed and/or preserved for histopathological examination.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 21 days of age.
- The P and F1 offspring selected for the post-weaning-developmental neurotoxicity were killed at Day 70 of age.
- All unselected offspring and those dying during the study were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
The following tissues were preserved from all F1 males and females from each dose group, in buffered 10% formalin: adrenals, coagulating gland, right epididymis, ovaries, right testis, pituitary, prostate, seminal vesicles, uterus with oviducts and cervix, vagina, gross lesions. The tissues from all control and high-dose animals and any treated animals which failed to mate or that did not achieve a pregnancy were processed. In addition, the following tissues were prepared for microscopic examination and weighed, respectively. The brain, spleen, thymus and uterus were weighed and preserved for one unselected male/female F1/F2 offspring at necropsy. In addition, samples of the following tissues of perfused animals at Day 70 of age (selected for the post-weaning-developmental neurotoxicity examinations) were histopathologically investigated: brain, dorsal root ganglia, dorsal and ventral root fibres, eyes, optic nerve, peroneal nerve, sciatic nerve, sural nerve, tibial nerve, skeletal muscle and spinal cord.

Statistics:

Data were initially assessed for homogeneity of variance using Levene´s test. Where variances were shown to be homogenous, a parametric assessment of the data was performed using one way analysis of variance (ANOVA), which if significant was followed by pairwise comparisons using Dunnett´s test. Where Levene´s test showed unequal variances, the data were analysed using non-parametric methodology: Kruskal-Wallis ANOVA which, if significant, was followed by Mann-Whitney U-test. Probability values (p) are presented as follows: p ≤ 0.001***, p ≤ 0.01**, p ≤ 0.05*, p > 0.05 (not significant).

Reproductive indices:

Mating performance and fertility
- Pre-coital interval: Calculated as the time elapsing between initial pairing and the observation of positive evidence of mating.

Fertility Indices
- Mating Index (%): (Number of animals mated/ number of animals paired) x 100
- Pregnancy Index (%): (Number of pregnant females/ number of females mated) x 100

Gestation and Parturition Data
- Gestation length: Calculated as the number of days of gestation including the day for observation of mating and the start of parturition. Where the start of parturition occurred overnight, the total was adjusted by adding half a day.
- Parturition Index (%): (Number of females delivering live offspring/ number of pregnant females) x 100

- Sex Ratio (% males): (Number of male offspring/ total number of offspring) x 100

Offspring viability indices:

Lactation Data
- Implantation losses (%):
% pre-implantation loss = [(Number of corpora lutea - number of implantation sites) / number of corpora lutea] x 100
% post-implantation loss = [(Group number of implantation sites - number of offspring) / number of implantation sites] x 100
offspring) x 100

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

25000 ppm: one P female was killed due to a decline in clinical condition; control/6000 ppm: one P female in each group was killed due to adverse clinical condition around the time of expected parturition, not treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1500/25000 ppm: increase in bodyweight gain during Days 1-21; 25000 ppm (P, female): significant lower food intake during the last week of lactation; 6000 ppm (P, female): higher food intake during last week of gestation and lactation, non-adverse.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1500/25000 ppm: increase in bodyweight gain during Days 1-21; 25000 ppm (P, female): significant lower food intake during the last week of lactation; 6000 ppm (P, female): higher food intake during last week of gestation and lactation, non-adverse.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

effects observed, treatment-related

Description (incidence and severity):

6000 ppm/control: one P female animal of each group showed an irregular cycle, non-adverse.

Reproductive function: sperm measures:

effects observed, treatment-related

Description (incidence and severity):

25000 ppm: lower homogenisation resistant spermatid counts (P amles) for the epididymides and testes, non-adverse.

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

25000 ppm: lower number of P females with live offspring, higher post-implantation loss, not treatment-related.

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no treatment-related deaths amongst parental animals. One female in the high-dose group showed dehydration, lethargy, emaciation, hunched posture, tip-toed gait and staining around mouth. Macroscopic necropsy examination indicated that the animal had a suspected broken jaw and the decline in clinical condition was considered to be unrelated to treatment. One control P female showed hunched posture, pilo-erection, tip-toed gait, lethargy pallor of the extremities and vaginal discharge on Day 97 of the P generation. One female in the mid-dose group showed hunched posture, pilo-erection, staining around the snout, staining around ano-genital region and pallor of the extremities on Day 99 of the P generation. These clinical signs coincided with the time of expected parturition and were considered to reflect difficulties with the birth of their litter. These animals were therefore killed for animal welfare considerations.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No adverse effects of treatment on mean bodyweight or bodyweight change was apparent in either generation for males and females. A significant increase in bodyweight gain during Days 1-21 in the low- and high-dose female P group was considered to be not treatment related.
No adverse effect of treatment on mean food consumption was apparent in males and females. Food consumption of P females in the high-dose group was significantly lower during the last week of lactation. In mid-dose P females a higher food intake was observed during the last week of gestation and lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
Achieved intakes for animals were in line with expectations throughout the P and F1 generations (see Table 1 under “Any other information on results incl. tables”).

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no obvious adverse effects of treatment on the oestrous cycles of females. One control and one mid-dose animal of the P generation showed an irregular cycle. There was no adverse effect of treatment on corpora lutea count, numbers of implantation sites or litter size at birth for females receiving the test substance. The mean number of small, medium or large oocyte follicles in the ovaries for F1 females of the high-dose group did not indicate any adverse effects of treatment (see Table 2 under “Any other information on results incl. tables”).

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No adverse effects of treatment on sperm concentration, motility or morphology were apparent for males receiving the test substance in either generation. For P males at the high-dose, homogenisation resistant spermatid counts for the epididymides and testes were lower than concurrent control, this decrease was considered to be incidental and of no toxicological significance (see Table 3 under “Any other information on results incl. tables”).

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no obvious adverse effects of treatment on mating performance, fertility or gestation length of females receiving the test substance. While the number of P females with live offspring was marginally lower than anticipated in the high-dose group, there was no similar occurrence in the following F1 generation and this finding was therefore considered coincidental and unrelated to treatment.
In total there were 26, 25, 26 and 23 P females at 0 (control), 1500, 6000 and 25000 ppm of the test substance, respectively, that successfully reared young to weaning. In P females at the high-dose group a higher post-implantation loss was observed (see Table 3 under “Any other information on results incl. tables”). There was no subsequent significant difference in litter size at birth for the P females at the high-dose and no similar increase in post-implantation loss for F1 females at this dosage. In view of this, the higher implantation loss observed was considered to be incidental and unrelated to treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
For P males receiving the mid-dose, absolute and bodyweight-relative organ weight for the left testis was significantly higher than control. In the absence of any significant increase for the right testis for these males or both testes for F1 males or any treatment related histopathological change, this effect was considered to be incidental and unrelated to treatment. For P females of the high-dose group, absolute and bodyweight-relative thyroid weights were significantly higher. There were no significant differences in thyroid weights for F1 females or for males in either generation at this inclusion level. Given the earlier and potentially higher exposure of the F1 females to the test substance (due to low bodyweight at this stage) and the absence of any accompanying effect on thyroid weights for these animals, this finding was therefore considered to be incidental and of no toxicological significance. For P females receiving the mid and high-dose, absolute uterine weight was significantly lower; values when adjusted for the bodyweight were not significantly different from control and this finding was therefore considered to be coincidental and to reflect normal biological variation. In the low-dose P female group bodyweight-relative brain weight was significantly lower than control. In the absence of any dosage relationship, and any effect on absolute brain weight, this was considered to be incidental and unrelated to treatment (see Table 5 under “Any other information on results incl. tables”).

GROSS PATHOLOGY (PARENTAL ANIMALS)
At 25000 ppm the brain of three P males were observed to be surrounded by clear fluid at necropsy, the brain form one of these animals was also misshapen. One control animal was also observed to have fluid surrounding the brain. In the absence of any evidence of treatment-related histopathological change or any similar findings for P females or for either sex in the F1 generation, this finding was considered to be incidental and unrelated to treatment. The majority of macroscopic necropsy findings of P females were limited to decedent animals; these deaths were considered to be unrelated to treatment.

HISTOPATHOLOGY (PARENTAL ANIMALS)
There were no histopathological findings observed for adults that were considered to indicate an adverse effect of the test substance at a dietary inclusion level of 25000 ppm.

POSITIVE CONTROL (PARENTAL ANIMALS)
Treatment at 10000 ppm DEPH was associated with clear signs of adult toxicity including notable effects on bodyweight and some organ weights. Reproduction of P animals was unaffected by treatment but shorter male ano-genital distance, increased male nipple counts and delays in attainment of sexual maturation suggested a possible endocrine disruption effect.

Dose descriptor:

NOAEL

Remarks:

toxicity/fertility

Effect level:

>= 25 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

NOAEL

Remarks:

toxicity/fertility

Effect level:

>= 1 159 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: mean achieved dose level (P males)

Dose descriptor:

NOAEL

Remarks:

toxicity/fertility

Effect level:

>= 2 200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: mean achieved dose level (P females)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

25000 ppm F1: litter weights were slightly lower; 1500/6000 ppm F2: bodyweight gain was slightly lower, non-adverse.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

1500 ppm F1 males and 6000 ppm F2 females: lower ano-genital distance; 25000 ppm F1 females: visible nipple counts marginally higher, non-adverse.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

25000 ppm female F1/F2: absolute and bodyweight-relative spleen weight decreased; 1500 ppm female F2: lower absolute and bodyweight-relative spleen weights; 6000 ppm female F1: bodyweight-relative uterus weights decreased, non-adverse.

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

VIABILITY (OFFSPRING)
Survival of the offspring to weaning was unaffected at the dietary concentrations by either maternal treatment or by more direct treatment when the offspring consumed the diet as they approached weaning in both generations.

CLINICAL SIGNS (OFFSPRING)
No adverse effects were observed for the test substance treated animals.

FOOD INTAKE (OFFSPRING)
Higher food intake for F1 females in the mid-dose group was observed during the last two weeks of gestation and Days 4-7 of lactation. F1 females in the low-dose group showed a significant higher food intake was also observed for the last week of gestation and the first week of lactation. In the absence of any consistent dosage relationship and the absence of any similar increases at the high-dose group, these differences were considered to be unrelated to treatment.

BODY WEIGHT (OFFSPRING)
There was no adverse effect of treatment on litter weights or bodyweight changes. For the F1 offspring at the high-dose litter weights were slightly lower between Days 14-21 (see Table 2 under “Any other information on results incl. tables”). For F2 offspring at the low and high-dose group bodyweight gain was slightly lower than control for both sexes between Day 7-14. These differences may reflect normal biological variation and were not considered to indicate an adverse effect of treatment.

SEXUAL MATURATION (OFFSPRING)
There were no obvious effects of treatment on the sexual maturation of either sex for the F1 animals receiving the test substance. Sex ratio at birth and subsequently at Day 21 of age was similar to concurrent control in all treatment groups over both generations. There was no adverse effect of treatment on mean ano-genital distance on Day 1 of age for either sex. For F1 males at the low-dose and F2 females at the mid-dose, mean ano-genital distance on Day 1 of age was slightly shorter (see Table 4 under “Any other information on results incl. tables”). These decreases were considered to reflect normal biological variation and were considered to be unrelated to treatment.
There was no adverse effect of treatment on visible nipple counts on Days 11-15 of age for either sex in either generation. Only for F1 females at the high-dose visible nipple counts were marginally higher than control only at Day 11 of age, therefore this effect was considered to be incidental and unrelated to treatment (see Table 6 under "Any other information on results incl. tables").

REPRODUCTIVE PERFORMANCE (OFFSPRING)
In total there were 21, 24, 22 and 24 pregnant F1 females at 0 (control), 1500, 6000 and 25000 ppm of the test substance, respectively, that successfully reared young to weaning. One control F1 and one mid-dose F1 female showed total litter loss post partum. As there was no consistency between the F0 and F1 generations and also no increase in mortality for offspring for those litters surviving to weaning in either generation, these isolated occurrences were considered to be incidental and unrelated to maternal treatment.

ORGAN WEIGHTS (OFFSPRING)
For female offspring receiving the high-dose, absolute and bodyweight-relative spleen weights were statistically lower than control in both generations. Statistically lower absolute and bodyweight-relative spleen weights were observed for F2 females at the low-dose. In the absence of any effect on spleen weight or any treatment related histopathological change for adult F1 females, this effect was considered to be of no long term toxicological significance. At the mid-dose, bodyweight-relative uterus weights of F1 offspring were significantly lower than control although absolute weights were not similarly affected in the absence of any effects for F2 offspring at this dosage, or any similar effect at the high-dose, this was considered to be incidental and unrelated to treatment. Assessment of intergroup differences in absolute and bodyweight-relative brain weights at Day 70 of age for offspring derived from dams receiving the test substance did not reveal any obvious adverse effects of previous maternal/pre-weaning exposure.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic abnormalities observed for both decedent and terminal kill offspring were typical for the age examined and neither the incidence or distribution of these findings indicated any adverse effect of treatment with the test substance in either generation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (OFFSPRING)
One high-dose animal of the F1 generation showed an irregular cycle.

POSITIVE CONTROL (OFFSPRING)
Adverse effects on offspring survival and growth were apparent and offspring failed to thrive following separation from the parent female at weaning. Effects on brain, spleen, thymus and, for females, uterus weight were apparent for the F1 offspring. Necropsy revealed enlarged liver and kidneys, small testes, epididymides and seminal vesicles for male F1 animals.

HISTOPATHOLOGICAL FINDINGS FOR BEHAVIOURAL OFFSPRING
There were no histopathological findings observed for male and female behavioural offspring that were considered to indicate an adverse effect at the high-dose level.

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

>= 25 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

>= 1 342 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: mean achieved dose level (F1 males)

Dose descriptor:

NOAEL

Remarks:

development

Generation:

F1

Effect level:

>= 2 262 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: mean achieved dose level (F1 females)

Reproductive effects observed:

not specified

Table 1. Overall achieved intakes during each generation for test material treated groups.

Dietary Concentration	Males (mg/kg bw/day)    F0   F1		Females (mg/kg bw/day) Maturation Gestation Lactation  F0     F1     F0      F1     F0    F1
1500 ppm	82	109	106	135	101	108	231	238
6000 ppm	324	435	431	540	411	434	919	918
25000 ppm	1159	1342	1392	1493	1665	1697	3544	3596

Table 2. Table for reproductive performance.

Parameter	Control group	Low-dose group 1500 ppm	Mid-dose group 6000 ppm	High-dose group 25000 ppm
Live Birth Index F0-F1 F1-F2	99.3 99.6	98.2 98.3	98.4 99.5	98.4 99.5
Pre-Implantation Loss [%] F0-F1 F1-F2	5.4 ± 4.6 5.5 ± 8.7	6.8 ± 7.4 4.9 ± 8.8	5.7 ± 4.0 5.4 ± 9.8	5.4 ± 6.1 1.7 ± 3.1
Post-Implantation Loss [%] F0-F1 F1-F2	3.1 ± 6.5 7.0 ± 8.7	3.7 ± 5.5 4.2 ± 5.2	5.0 ± 7.4 4.9 ± 7.8	10.8 ± 16.2** 7.7 ± 13.8
Total Litter Weight (g) F1 Day 1 F1 Day 14 F1 Day 21 F2 Day 1 F2 Day 14 F2 Day 21	95.1 389.5 600.0 87.3 363.2 564.2	89.6 362.6 567.4 101.0 384.1 599.3	92.4 368.8 584.8 95.6 368.9 570.4	88.9 343.7** 523.8** 98.0 360.0 555.2
Total number of Corpora Lutea F0-F1 F1-F2	15.7 ± 1.6 15.3 ± 2.3	15.7 ± 2.7 17.2 ± 2.2	15.7 ± 1.8 16.5 ± 2.9	15.9 ± 2.0 16.3 ± 3.8
Total number of Implantation Sites F0-F1 F1-F2	14.8 ± 1.7 14.4 ± 2.5	14.5 ± 2.7 16.3 ± 1.8	14.8 ± 2.0 15.4 ± 2.4	15.0 ± 1.7 16.0 ± 3.7
Total Number of Offspring Born F0-F1 F1-F2	14.4 ± 2.1 13.5 ± 2.9	14.0 ± 2.6 15.5 ± 1.7	14.2 ± 2.4 14.6 ± 2.5	13.4 ± 1.7 14.8 ± 4.0
Sex ratio (% male at birth)	51.9	45.3	46.8	51.8

p** ≤ 0.01

Table 3. Results of sperm characterisation.

Parameter	Control group	High-dose group 25000 ppm
Sperm concentration [M/mL] F0 F1	92.2 107.0	102.5 107.0
Motility (%) F0 F1	52 58	57 58
Sperm Morphology, Number(%) F0 F1	99.9 100.0	99.8 100.0

Table 4. Ano-Genital distance of offspring - group mean litter values.

Ano-genital distance (mm) on Day 1 post partum	Control group	Low-dose group 1500 ppm	Mid-dose group 6000 ppm	High-dose group 25000 ppm
Males F1 F2	3.12 ± 0.34 3.01 ± 0.33	2.95* ± 0.40 3.03 ± 0.33	3.12 ± 0.46 3.01 ± 0.33	3.13 ± 0.36 3.11 ± 0.31
Females F1 F2	1.60 ± 0.34 1.47 ± 0.19	1.65 ± 0.44 1.51± 0.24	1.49 ± 0.31 1.33 ± 0.14**	1.67 ± 0.34 1.49 ± 0.22

p** ≤ 0.01

Table 5. Organ weights.

Absolute organ weights – group mean values
Organ	Control group	Low-dose group 1500 ppm	Mid-dose group 6000 ppm	High-dose group 25000 ppm
Left testes F0 males F1 males	1.8398 ± 0.2431 1.8445 ± 0.1758	1.9042 ± 0.1890 1.8268 ± 0.1048	1.9683 ± 0.1463* 1.8508 ± 0.1759	1.9484 ± 0.1093 1.8158 ± 0.1950
Thyroid females F0 F1	0.0168 ± 0.0036 0.0191 ± 0.0042	0.0170 ± 0.0032 0.0184 ± 0.0053	0.0167 ± 0.0036 0.0182 ± 0.0033	0.0212± 0.0044*** 0.0182 ± 0.0045
Uterus females F0 F1	0.5181 ± 0.1530 0.5396 ± 0.1622	0.4434 ± 0.112 0.5563 ± 0.1649	0.4640 ± 0.2018* 0.5428 ± 0.1649	0.4291 ± 0.0856* 0.5226 ± 0.1183
Organ weights for female offspring – group mean values
Spleen absolute F1 F2 Spleen. relative F1 F2	0.1861 ± 0.0576 0.1868 ± 0.0646   0.4226 ± 0.0615 0.4096 ± 0.0799	0.1644 ± 0.0526 0.1451 ± 0.0443*   0.3899 ± 0.0879 0.3507 ± 0.0527*	0.1642 ± 0.0589 0.1534 ± 0.0493   0.3762 ± 0.0764 0.3656 ± 0.0713	0.1485 ± 0.0513* 0.1370 ± 0.0590**   0.3553 ± 0.0719** 0.3257 ± 0.0748***
Uterus absolute F1 F2 Uterus relative F1 F2	0.0923 ± 0.0265 0.0539 ± 0.0113   0.2111 ± 0.0322 0.1210 ± 0.0158	0.0877 ± 0.0265 0.0536 ± 0.0182   0.2119 ± 0.0601 0.1324 ± 0.0426	0.0831 ± 0.0274 0.0513 ± 0.0212   0.1931 ± 0.0456* 0.1225 ± 0.0348	0.1242 ± 0.1746 0.0517 ± 0.0242   0.2967 ± 0.4169 0.1276 ± 0.0489

p* ≤ 0.05, p** ≤ 0.01, p*** ≤ 0.001

Table 6. Nipple counts for the F1 generation at Day 11 of age.

Group	Mean Nipple count at Day 11 of age
	Male	Female
Control	0.0 ± 0.0	9.4 ± 1.8
Low dose group	0.1 ± 0.5	10.4 ± 1.5
Mid-dose group	0.0 ± 0.0	9.9 ± 1.5
High-dose group	0.1 ± 0.3	10.6 ± 1.4*

p* ≤ 0.05

Conclusions:

The test substance had no effect on reproductive performance.

Reference 4

Endpoint:

fertility, other

Remarks:

other: effects on reproductive organs

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with acceptable restrictions. Report does not contain all study details. Female fertility data are identical in rats and mice, indicating a transcription error.

Principles of method if other than guideline:

Within a 90 day oral feeding study performed equivalent to OECD guideline 408 with Castor oil, male and female fertility parameters were analyzed in rats and mice. No matings were performed.

GLP compliance:

yes

Limit test:

no

Species:

other: rats and mice

Strain:

other: F344/N and B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Simonsen Laboratories (Gilroy, CA, USA)
- Age at study initiation: 6 weeks
- Weight at study initiation: male rats: 126 - 132 g; female rats: 107- 110 g, male mice: 22.6 - 23.0 g, female mice: 17.2 - 17.7 g
- Fasting period before study:
- Housing: rats: 5 per cage, mice individually in Polycarbonate cages lined with heat-treated hardwood chips, covered with polyester filter sheets.
- Diet: Control feed (NIH 07) or diet formulations of castor oil were available ad libitum; feeders were changed twice per week throughout the study.
- Water: automatic watering system, ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-76
- Humidity (%): 42-72
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulated diets were prepared by blending the appropriate amount of castor oil with a small quantity of feed to prepare a premix. The premix then was layered between the required amount of feed in a twin-shell blender and blended for 15 minutes to achieve a uniform mix. The homogeneity of castor oil in feed at 10% (100 mg/g) was determined by gravimetric analysis, and blends at 0.5% (5 mg/g) were determined by HPLC analysis. These concentrations of chemical in feed were found to be homogeneously distributed by this mixing procedure. The stability of the 0.5% dose level was determined using HPLC; it was found to be stable for at least 21 days when stored in the dark at 5°C and for 3 days when stored open to air and light in a rodent cage. During the studies, formulated diets were stored for no longer than 3 weeks at 5°C; feed hoppers in the animal cages were changed twice weekly.

Details on mating procedure:

no matings performed

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The results of the analyses for all dose mixtures given to the animals ranged from 97% to 106% of the target concentrations.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily ad libitum feeding

Remarks:

Doses / Concentrations:
0, 2.50, 5.00, 10.0 % (w/w)
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
0, 1583, 3067 and 5835 mg/kg bw/day
Basis:
other: actual ingested: male rats

Remarks:

Doses / Concentrations:
0, 1569, 3045, 5725 mg/kg bw/day
Basis:
other: actual ingested: female rats

Remarks:

Doses / Concentrations:
0, 3800, 7823, 15017 mg/kg bw/day
Basis:
other: actual ingested: male mice

Remarks:

Doses / Concentrations:
0, 5009, 9627, 16786 mg/kg bw/day
Basis:
other: actual ingested: female mice

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS/ CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: initially and 1 x wk thereafter.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

Oestrous cyclicity (parental animals):

Proestrus, Estrous, Metestrous, Diestrous, Cycle Length (days)

Sperm parameters (parental animals):

Caudal weight, epididymal weight, testis weight, Sperm countig testis, Sperm motility (%)

Litter observations:

not performed

Postmortem examinations (parental animals):

Complete histopathology examinations were conducted on all rats and mice from the control and 10% dose groups. Livers were examined from male rats in all other dose groups; histologic sections of gross lesions were examined from all rats. Organ weights were determined to the nearest milligram for the liver, right kidney, right testicle, heart, thymus, and lungs. All tissues were preserved in 10% neutral buffered formalin.

The following tissues were routinely processed for preparation of histologic sections and microscopic examination: adrenal glands, brain, cecum, colon, duodenum, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, esophagus, eyes (if grossly abnormal), femur (including marrow), heart, ileum, jejunum, kidneys, liver, lungs and mainstem bronchi, mammary gland, mandibular and mesenteric lymph nodes, nasal cavity and turbinates, pancreas, parathyroid glands, pituitary gland, preputial or clitoral glands, rectum, salivary glands, skin, spinal cord and sciatic nerve (if neurologic signs present), spleen, forestomach and glandular stomach, thymus, thyroid gland, trachea, urinary bladder, zymbal glands, and all gross lesions and tissue masses including regional lymph nodes. A complete histopathologic examination was conducted on all rats and mice from the control and 10% dose groups. Liver was examined from male rats in all other dose groups, and histologic sections of gross lesions were examined from all rats.

Postmortem examinations (offspring):

not performed

Statistics:

Dunn's test; Shirley's test.

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:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

CLINICAL SIGNS AND MORTALITY
No effects

BODY WEIGHT AND WEIGHT GAIN
Group mean body weights of rats receiving diets containing castor oil did not differ significantly from controls. Mean body weights of exposed female rats were slightly lower than the mean body weights of controls but the differences were not dose-related.
Mean body weights of exposed male mice generally were lower than controls, while mean body weights of exposed females generally were higher. There were no obvious indications that these differences were related to dietary concentrations of castor oil, except that mean body weights of male mice receiving the 10% castor oil diet were consistently lower than those of control mice from week 3 through the end of the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No significant differences in average food consumption among each sex were observed, although food consumption of male and female rats receiving diets containing 10% castor oil was slightly lower than that of controls. Similarly, food consumption by female mice receiving diets containing 10% castor oil was slightly lower than controls.

ORGAN WEIGHTS
In male rats, there was a slight decrease in epididymal weight (6-7%) which occurred in the middle- and high-dose groups, but this was not dose-related. There were no effects on any other male rat reproductive endpoint, or on any female rat reproductive endpoint. Although there was some variation in epididymal weights, their small magnitude and the absence of changes in other endpoints suggested that there was little or no evidence of any reproductive toxicity associated with castor oil exposure. Histopathologic examination revealed an absence of compound-related lesions in any organ or tissue of rats exposed to castor oil in the diet.

Castor oil exposure produced no adverse effects on any male (testes weight, epididymal sperm motility, density, or testicular spermatid head count) or female (estrual cycle length, or time spent in each phase of the cycle) reproductive parameter among mice. The low value for sperm motility
in control mice was attributed to poor preparative technique.

Dose descriptor:

NOAEL

Remarks:

parental fertility parameters

Effect level:

ca. 5 000 mg/kg bw/day (actual dose received)

Based on:

other: calculated test material intake based on food consumption and body weight

Sex:

male/female

Basis for effect level:

other: for rats based on oestrus stage and cycle length and sperm characterization.

Dose descriptor:

NOAEL

Remarks:

parental fertility parameters

Effect level:

ca. 15 000 mg/kg bw/day (actual dose received)

Based on:

other: calculated test material intake based on food consumption and body weight

Sex:

male/female

Basis for effect level:

other: for mice based on oestrus stage and cycle length and sperm characterization.

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

not examined

Dose descriptor:

NOAEL

Generation:

F1

Based on:

not specified

Sex:

not specified

Remarks on result:

not measured/tested

Remarks:

/fertility study

Reproductive effects observed:

not specified

Table 1: Reproductive System Data for F344/N Rats in the 13-Week Feed Studies

of Castor Oil

 

	Percent in Feed
	0	2.5	5	10
Male^a
Left caudal weight (mg)	151	153	145	153
Left epididymal weight (mg)	502	498	464*	476
Left testis (mg)	1539	1550	1463	1492
Sperm count (x10^6)/g testis	72.8	65.9	71.7	77.5
Sperm motility (%)	73.6	65.9	72.1	69.8
Female^b
Estrous stage (%)
Proestrus	12.5	14.2	15.8	16.7
Estrous	28.3	32.5	25.8	25.8
Metestrous	18.3	19.2	18.3	19.2
Diestrous	40.8	34.2	39.2	38.3
Not clear or no cells observed	0.0	0.0	0.8	0.0
Cycle Length (days)	5.0	5.1	5.2	5.1

^aMean for groups of 10 animals; no significant difference vs. the controls by Dunn's test

^bMean for groups of 10 animals unless otherwise specified

* Significantly different from control groups by Shirley's test ; p < 0.05.

 

Table 2: Reproductive System Data for B6C3F1 Mice in the 13-Week Feed Studies

of Castor Oil

 

	Percent in Feed
	0	2.5	5	10
Male^a
Left caudal weight (mg)	15	13	16	16
Left epididymal weight (mg)	45	46	46	44
Left testis (mg)	121	120	121	119
Sperm count (x106)/g testis	179.2	162.4	170.1	158.3
Sperm motility (%)	39.2	53.7	45.4	52.2
Female^b
Estrous stage (%)
Proestrus	12.5	14.2	15.8	16.7
Estrous	28.3	32.5	25.8	25.8
Metestrous	18.3	19.2	18.3	19.2
Diestrous	40.8	34.2	39.2	38.3
Not clear or no cells observed	0.0	0.0	0.8	0.0
Cycle Length (days)	5.0	5.1	5.2	5.1

^aMean for groups of 10 animals; no significant difference vs. the controls by Dunn's test

^bMean for groups of 10 animals unless otherwise specified

 

Reference 5

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

17 Feb - 07 April 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Crl:WI(Han) (outbred, SPF-Quality)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle Cedex, France
- Age at study initiation: approximately 12 weeks
- Weight at study initiation: 277-353 g (males), 180-228 g (females)
- Housing : 5 animals/sex/cage in Macrolon cages (MIV type, height 18 cm). This was also applicable for Main females and Recovery animals throughout the complete treatment period. Sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 (actual range: 19.7-21.9)
- Humidity (%): 40-70 (actual range: 22-71)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

This species and strain of rat has been recognized as appropriate for general and reproduction toxicity studies. The testing laboratory has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6h prior to dosing and were homogenized to a visually acceptable level.
Adjustment was made for the density of the test substance (0.944), and the specific gravity of the vehicle (1.125).

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at the testing laboratory.
- Purity: polyethylene glycol 400, specific gravity 1.125 (Merck, Darmstadt, Germany)

Dose volume: 5 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: max. 14 days
- Proof of pregnancy: evidence of sperm in the vaginal lavage, by staging of the estrous cycle and/or by the appearance of an intravaginal copulatory plug referred to as Day 0 of pregnancy
- After successful mating each pregnant female was caged individually in Macrolon cages (MIII type).

Following a minimum of 14 days of exposure for the males and females, one Repro female was cohabitated with one Main male of the same treatment group, avoiding sibling mating (Charles River supplied non-litter mates). Detection of mating was not confirmed for animal no. 98 which did deliver. The mating date of this animal was estimated at 21 days prior to the actual delivery date. This day was designated Day 0 post-coitum. Once mating had occurred, the males and females were separated. A maximum of 14 days was allowed for mating. After 14 days of mating, females who had not shown evidence of mating were separated from their males.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability. Samples of formulations were analysed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6h at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% for suspensions. Homogeneity was demonstrated if the coefficient of variation was ≤10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10%) and formulations at the entire range were stable when stored at room temperature for at least 6h.

Duration of treatment / exposure:

41-49 days,
i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Frequency of treatment:

daily, 7 days/week

Details on study schedule:

Offspring were euthanized at the age of 4 days.

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 10-day dose range finding study

Parental animals: Observations and examinations:

Male number paired with, mating date, confirmation of pregnancy, and delivery day were recorded.
Pregnant females were examined to detect signs of difficult or prolonged parturition, and cage debris of these females was examined to detect signs of abortion or premature birth. Any deficiencies in maternal care (such as inadequate construction or cleaning of the nest, pups left scattered and cold, physical abuse of pups or apparently inadequate lactation or feeding) were examined.

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1

Oestrous cyclicity (parental animals):

Uterus epithelium was analysed histologically for estrus, proestrus and cystic endometrial glands.

Sperm parameters (parental animals):

Parameters examined in [all/P/F1/F2] male parental generations: testis weight, epididymis weight, histology of testes
Of the first 5 Main males of the control and high dose group, additional slides of the testes were prepared to examine staging of spermatogenesis.
The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin.

Litter observations:

On Day 1 of lactation, all pups were randomized per litter and individually identified by means of subcutaneous injection of Indian ink.
Each litter was examined to determine the following, if practically possible:
Mortality / Viability: The numbers of live and dead pups on Day 1 of lactation and daily thereafter were determined. If possible, defects or cause of death were evaluated.
Clinical signs: At least once daily, detailed clinical observations were made in all animals.
Body weights: Live pups were weighed on Days 1 and 4 of lactation.
Sex: Sex was determined for all pups on Days 1 and 4 of lactation.

Postmortem examinations (parental animals):

SACRIFICE
- Maternal animals which delivered: on lactation Day 5
- Maternal animals which failed to deliver (4 animals): Post-coitum Day 26 (females with evidence of mating) or approximately 21 days after the last day of the mating period (females without evidence of mating).

GROSS NECROPSY
- Gross necropsy consisted of: see details under 7.5.1
The numbers of former implantation sites and corpora lutea were recorded for all paired females.

HISTOPATHOLOGY / ORGAN WEIGHTS
see details under 7.5.1

Postmortem examinations (offspring):

SACRIFICE
- Pups surviving to planned termination were killed by decapitation on lactation Day 5.

All pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk. If possible, defects or cause of death were evaluated. Any abnormal pup, organ or tissue as well as pups from females that were killed in extremis were preserved in 10% buffered formalin for possible further examination.

Statistics:

See Section 7.5.1

Reproductive indices:

For each group the following calculations were performed:
- Mating (%): Number of females mated/Number of females paired x 100
- Fertility index (%): Number of pregnant females/Number of females paired x 100
- Conception index (%): Number of pregnant females/Number of females mated x 100
- Gestation index (%): Number of females bearing live pups/Number of pregnant females x 100
- Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive x 100

Offspring viability indices:

For each group the following calculations were performed:
- Percentage live males at First Litter Check: Number of live male pups at First Litter Check/ Number of live pups at First Litter Check x 100
- Percentage live females at First Litter Check: Number of live female pups at First Litter Check/Number of live pups at First Litter Check x 100
- Percentage of postnatal loss Days 0-4 of lactation: Number of dead pups on Day 4 of lactation/Number of live pups at First Litter Check x 100
- Viability index (%): Number of live pups on Day 4 of lactation/Number of pups born alive 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 specified

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

as found by histological examination of uterus

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

as found by histological examination of testes

Reproductive performance:

no effects observed

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The assessment of the integrity of the spermatogenetic cycle did not provide any evidence of impaired spermatogenesis, as explored by histological examination of testes.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Histological examination of the uterus epithelium and endometrial glands did not reveal any treatment related influences on estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment related effects on reproductive parameters were noted.
The mating, fertility and conception indices, precoital time, and number of corpora lutea and implantation sites were unaffected by treatment.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No treatment-related effects observed.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related effects observed.

For further details see 7.5.1

Dose descriptor:

NOAEL

Remarks:

parental fertility

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

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:

no effects observed

Number of pups: 432
No toxicologically relevant effects on gestation index and duration, parturition, maternal care and early postnatal pup development (mortality, clinical signs, body weight and macroscopy) were observed.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

other: macroscopy

Reproductive effects observed:

not specified

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

An 28 day oral gavage screening study (van Otterdijk, 2010) was performed according to OECD guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test) in Crl:WI(Han) (outbred, SPF-Quality) Wistar Han rats with Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052 -13 -0). Doses of 0, 100, 300 and 1000 mg/kg bw/d were given to groups of four Main groups of 10 male and 5 female rats. Additionally, 5 Recovery group males and females in the control and high dose group were allowed 14 days of recovery.

An additional 10 females were added to each group for the assessment of reproduction and developmental toxicity. Recovery animals were exposed for at least 28 days from start of treatment up to termination or start of recovery. Females used for the assessment of reproduction/developmental toxicity were exposed for 41-49 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation.

Gonadal function was examined by histological evaluation of reproductive organs. Mating behavior, conception, parturition, clinical signs, mortality, body weight, food consumption, gross pathology, organ weights, histopathology, mating index, fertility index, number of implantation sites, duration of pregnancy, birth index, live birth index, pregnancy index, litter size, litter weight, pup weight, sex ratio, survival index, viability index were determined for all dose groups. No treatment related abnormalities were observed. Therefore a NOAEL for parental fertility of 1000 mg/kg bw/d was found.

Structural analogue read-across from Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052-13-0) for mammalian toxicity was judged to be justified for the following reasons: This read across substance is also a glyceride, containing mainly C12-14 fatty acid and actetate moiety as well as glycerol. It¿s an organic liquid with a pour point of -8 °C and a melting point of 357.85 °C and a vapour pressure < 0.01 Pa at room temperature. In contrast to the glycerides of the fatty acid glyceride category, it has a higher water solubility of 8.75 mg/L, which might influence its environmental distribution, but not the mammalian metabolism upon systemic uptake. Therefore it is expected to feed into the same mammalian physiological pathways as the members of the fatty acid glyceride category, like citric acid cycle, sugar synthesis and lipid synthesis. These processes are described in detail within the category justification.

A 90 day oral feeding study with Castor oil (CAS No. 8001 -79 -4) was performed equivalent to OECD Guideline 408 in F344/N rats and B6C3F1 mice (Irwin, NTP report 1992). The test substance was mixed at concentrations of 0, 0.62, 1.25, 2.50, 5.00, 10.0 % (w/w) to the diet and the animals were fed ad libitum for 13 weeks. 10 animals per sex and per dose were used. The highest dose was equivalent to approx. 5.7 g/kg bw/day for rats and approx. 15 g/kg bw/day for mice. No matings were performed, but male and female fertility parameters were analyzed in rats and mice including oestrous cycle length, caudal weight, epididymal weight, testis weight, sperm count/g testis, sperm motility (%) and histopathology of organs relevant for reproduction (including adrenal glands, epididymis/seminal vesicles/prostate/testes or ovaries/uterus, mammary gland, pituitary gland, preputial or clitoral glands). A complete histopathologic examination was conducted on all rats and mice from the control and 10% dose groups.

No significant changes were noted in a screening for male reproductive endpoints, including sperm count and motility, and no changes were observed in the length of oestrous cycles of rats or mice given diets containing castor oil. No histopathologic abnormalities were found in the reproductive organs.

A NOAEL of 5000 mg/kg bw/day for rats and a NOAEL of 15000 mg/kg bw/day for mice could be identified based on parental fertility parameters.

Effects on developmental toxicity

Description of key information

All available studies on developmental toxicity and teratogenicity of Glycerides resulted in NOAELs ≥ 1000 mg/kg bw/day:

Rat: NOAEL (F1/F2 ≥ 1342/2262 mg/kg bw/day (OECD 416/426)

Rabbit: NOAEL = 1000 mg/kg bw/day (similar to OECD 414)

Mouse: NOAEL = 9540 mg/kg bw/day (teratogenicity study)

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the same study as described above (van Otterdijk, 2010) with Glycerides, C8-18 and C18-unsatd. mono- and di-, acetates (CAS No. 91052 -13 -0) a total number of 432 pups was euthanized at the age of 4 days. Based on litter size and weights, number viable (number alive and number dead), sex ratio, postnatal growth, postnatal survival, grossly visible external and soft tissue abnormalities a developmental NOAEL of 1000 mg/kg bw/d was found for Wistar rats.

A developmental toxicity study with rats and rabbits was conducted with a 20% lipid emulsion containing a 3:1 ratio of MCT (Medium Chain Triglycerides):LCT (Long Chain Triglycerides) (Henwood, 1997). Doses of 1000 and 4280 mg/kg bw/d were given intravenously from gestation day 6 through 15 (rats) or GD 7 through 19 (rabbits). The intravenous route of administration was used because the lipid emulsion is intended for intravenous human administration as a component of parenteral nutrition. The dose was administered daily to rats by intravenous infusion via a caudal vein and to rabbits via a marginal ear vein. In rats there were no treatment related direct teratogenic effects observed. In rabbits administration of the test article resulted in lower maternal food consumption and significant body weight loss during treatment at the highest dose level. Therefore, the observed foetal effects (i.e., increased resorptions, decreased fetal body weights, and increased incidence of morphological anomalies) were assumed to be the result of dietary deprivation, maternal toxicity, or both, rather than a direct teratogenic effect of the test article. The NOAEL was therefore set to be 4280 mg/kg bw /day for developmental toxicity for both, rats and rabbits.

Justification for classification or non-classification

According to DSD (67/548/EEC) or CLP (1272/2008/EC) classification criteria for reproduction, no classification is required.