2-methoxyethyl acrylate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Jan - 23 Mar 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Food and Consumer Product Safety Authority (VWA), GG Utrecht, The Netherlands

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-methoxyethyl acrylate

EC Number:

221-499-3

EC Name:

2-methoxyethyl acrylate

Cas Number:

3121-61-7

Molecular formula:

C6H10O3

IUPAC Name:

2-methoxyethyl acrylate

Details on test material:

- Name of test material (as cited in study report): 2-methoxyethyl acrylate
- Physical state: clear colourless liquid
- Analytical purity: ≥ 99%
- Lot/batch No.: C211114SP6
- Expiration date of the lot/batch: 2012-06-12
- Stability under test conditions: stable
- Storage condition of test material: at room temperature (< 35 °C) in the dark in a well-sealed container
- Other: study specific test substance information: hygroscopic and volatile (22.7 hPa at 61 °C)

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: (P) 11 wks
- Weight at study initiation: (P) Males: 255-301 g; Females: 179-205 g
- Housing: during pre-mating, animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages (MIV type, height 18 cm). During mating, females were caged together with males on a one-to-one-basis in Macrolon plastic cages (MIII type, height 18 cm). After mating, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm). During lactation, pups were kept with the dam until termination in Macrolon plastic cages (MIII type, height 18 cm). During locomotor activity monitoring of the dams, the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes. Generally, sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment/nesting material (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment or bedding material.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0-23.7
- Humidity (%): 33-95
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 Jan 2012 To: 23 Mar 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: test substance formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and test substance. No correction was made for purity of the test substance. Formulations were stored at room temperature (< 35 °C) in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on solubility
- Concentration in vehicle: 7.72, 19.3 and 29% for dose levels of 40, 100 and 250/150 mg/kg bw/day, respectively
- Amount of vehicle (if gavage): 5 mL/kg bw

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 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: individually in Macrolon plastic cages (MIII type, height 18 cm).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations at target concentrations of 7.72, 19.3 and 29% were analysed for analytical concentrations using HPLC and UV detection at 205 nm. Homogeneity and stability were assessed in the highest and lowest dose formulation (7.72 and 29%) using the same analytical method (HPLC/UV). The analytical concentrations determined in all dose formulations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The highest and lowest dose formulations were homogenous (i.e. coefficient of variation ≤ 10%). After storage, the highest and lowest dose formulations yielded a relative difference of ≤ 10%. Based on these results, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

P (Males): 31-35 days, i.e. 2 weeks prior to mating, during mating, and up to termination
P (Females): 42-56 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, 7 days/week

Details on study schedule:

- Age at mating: ca. 13 weeks

Doses / concentrations

Remarks:

Doses / Concentrations:
40, 100 and 250/150 mg/kg bw/day (250 mg/kg bw/day: from Day 1 to 11; 150 mg/kg bw/day: from Day 12 to study termination)
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: dose levels were based on a preliminary range finding study, in which 3 female rats per dose group received the test substance at 100, 200 and 400 mg/kg bw/day. Animals treated with 100 and 200 mg/kg bw/day received the test substance for a period of 10 days, whereas animals of the 400 mg/kg bw/day group were only treated for 2 days. On Day 2 of the study, one animal died and piloerection was observed in one animal at 400 mg/kg bw/day. At necropsy, abnormalities of the stomach, lungs and thymus were found in the animals of this dose group. No mortalities occurred at 100 and 200 mg/kg bw/day. At both dose levels, salivation was observed in all animals during the first days of the study (Day 1-3). No adverse changes in body weights and food consumption were observed and macroscopic examination did not reveal any abnormal findings, except for a reduced thymus size in all animals treated with 200 mg/kg bw/day. Based on the results of this range finding study, dose levels for the main study were 40, 100 and 250 mg/kg bw/day.
- Other: from Day 12 of study onwards, the dose level was lowered to 150 mg/kg bw/day for both sexes due to severe toxicity noted at 250 mg/kg bw/day.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed for mortality at least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: detailed clinical observations were made daily in all animals, at least immediately after dosing (on the peak period of anticipated effects after dosing). Once prior to start of treatment and at weekly intervals during the treatment period observations were also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed sign was recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were determined on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and during lactation on Days 1 and 4. In order to monitor the health status, animals treated at 250/150 mg/kg bw/day were weighed more often.

FOOD CONSUMPTION: food consumption was determined weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Sperm parameters (parental animals):

Parameters examined in P male parental generations:
testis weight, epididymis weight, other: weight of seminal vesicles including coagulating glands

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

GROSS EXAMINATION OF DEAD PUPS:
no, 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, after completion of the mating period
- Maternal animals: all surviving animals which delivered, on lactation Days 5-7
Females which did not deliver were sacrificed on post-coitum Days 25-27 (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 external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 and 2 under “Any other information on materials and methods incl. tables” were prepared for microscopic examination and weighed, respectively. Staging of spermatogenesis in testes was performed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed on Days 5-7 of lactation.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: all pups were sexed and descriptions of all external abnormalities were recorded. The stomach was examined for the presence of milk.

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Reproductive indices:

Mating index (%) = (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 pubs/number of pregnant females) x 100
Duration of gestation = number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

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 post-partum/number of pups born alive) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

250 mg/kg bw/day: 2 males died on Day 2, 1 male was killed on Day 8; 100 mg/kg bw/day: 1 female killed; detailed clinical signs see “Results of examinations: parental animals”

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

250 mg/kg bw/day: body weight loss; 250/150 mg/kg bw/day: body weight loss during first 2 weeks of post-coitum and during the last week (f); 100 mg/kg bw/day: body weight loss at end of post-coitum (f)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

250 mg/kg bw/day: body weight loss; 250/150 mg/kg bw/day: body weight loss during first 2 weeks of post-coitum and during the last week (f); 100 mg/kg bw/day: body weight loss at end of post-coitum (f)

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

abnormal findings in stomach, testes, epididymides, spleen, liver, uterus, mammary gland, thymus: see “Results of examinations: parental animals”

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

40 and 250/150 mg/kg bw/day: increase in precoital time (f); 250/150 mg/kg bw/day; decreased fertility and conception indices (only 2 of 9 animals pregnant); 100 and 250 mg/kg bw/day: decrease in number of corpora lutea and implantation sites (f)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
-Unscheduled deaths: at 250 mg/kg, severe parental toxicity was noted. Toxicity consisted of two male animals found dead on Day 2 of study (no cause of death could be determined) and one male animal that was killed in extremis on Day 8 of study (showed ulcerative inflammation in the stomach with resultant peritonitis). At 100 mg/kg bw/day, one female was killed in extremis on Day 21 post-coitum. Clinical signs in males and females found dead at both dose levels included hunched posture, piloerection, pale and lean appearance.
- Scheduled deaths: treatment-related clinical signs were noted at 250 mg/kg bw/day, and consisted of hunched posture (18 animals 1-5 days), piloerection (1 female 2 days), salivation (3 animals 1 day), and rales (1 female 1 day). These findings disappeared during dosing at 150 mg/kg bw/day. Red vagina or bleeding from the vagina was noted for three females treated at 200 and 250/150 mg/kg bw/day on Days 14, 19 and 23 post-coitum, respectively. This was probably bleeding caused by loss of fetuses and considered treatment-related. These females showed total litter loss or implantation sites only. One female treated at 150 mg/kg bw/day showed hunched posture and piloerection on Days 19 to 21 post-coitum. As this was not noted for the other females at this dose, and recovered during further treatment, it was not considered toxicologically significant.
Incidental findings that were noted included salivation, alopecia, scales and scabs. These findings occurred within the range of background findings to be expected for rats of this age and strain and were thus considered to be of no toxicological relevance.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
At 250 mg/kg bw/day, most male animals and a few female animals showed a body weight loss, which slightly recovered during treatment at 150 mg/kg bw/day. Reduced body weight gains were also noted for males at 100 mg/kg bw/day. At 250/150 mg/kg bw/day, reduced body weight gains were observed during the first two weeks of post-coitum and a body weight loss during the last week. A body weight loss was also noted at the end of postcoitum for females treated with 100 mg/kg bw/day. The reduced body weight gains for females of the 100 mg/kg bw/day dose group during the first two weeks of post-coitum was considered a cause of their pregnancy status (i.e. implantation sites only instead of live fetuses) and not considered toxicologically relevant. No changes in body weights were observed at 40 mg/kg bw/day. Since no litters were born at the 100 and 250/150 mg/kg bw/day, no body weight gain of these animals during lactation could be determined.
Reduced food consumption was noted for the first two weeks of treatment at 250 mg/kg bw/day for both sexes. This recovered during treatment at 150 mg/kg bw/day. At the end of post-coitum, a dose related decrease in food consumption was noted for females treated at 100 mg/kg bw/day (Days 17-20) and 250/150 mg/kg bw/day (Days 14-20) when compared to the concurrent control group. The lower food consumption levels at 100 mg/kg bw/day (Days 7 to 17 post-coitum) and 250/150 mg/kg bw/day (Days 7-14 post-coitum) were considered a cause of their pregnancy status (i.e. implantation sites only or not pregnant), and therefore not considered toxicologically relevant. During lactation, females treated at 40 mg/kg bw/day showed reduced food consumption when compared to the concurrent control animals. However, this was considered due to the reduced number of live pups in these litters. No data on food consumption during lactation was obtained for 100 and 250/150 mg/kg bw/day as no litters were born in these groups. All other statistical significant changes were not considered toxicologically relevant as the values were within normal limits.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Estrous cyclicity was not determined in females. However, there was evidence of cyclic changes in the reproductive tracts of non-pregnant females treated with 250/150 mg/kg bw/day.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Staging of spermatogenesis in testes provided evidence of test article related impairment to the spermatogenetic cycle at all dose levels in a dose related manner. The most subtle changes were those of enlarged spermatagonia with finely granular cytoplasm and asynchronous tubules in which normal cell associations were absent. Individual cell necrosis, spermatidic giant cells, and reduced layers of spermatagonia were common observations. At the highest 250/150 mg/kg bw/day dose level there were no recognizable stages. Tubules were often lined by a single layer of cells with uncertain identity (Sertoli cells, primary spermatagonia or both). Mitotic figures were occasionally present and appeared abnormal.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): see Table 3 under “Any other information on results incl. tables”)
Male and female reproduction was affected at all dose levels. Precoital time of females treated at 40 and 250/150 mg/kg bw/day was increased. Two females of the high dose group did not mate in the first pairing period of 15 days. Out of these, one female mated with her second male, whereas the other did not. Fertility and conception indices were decreased at 250/150 mg/kg bw/day, as out of the nine mated females only two were pregnant. In addition, a dose related decrease was noted for number of corpora lutea and implantation sites at 100 and 250/150 mg/kg bw/day. At 40 mg/kg bw/day, an increased duration of gestation was noted.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In males treated at 100 and 250/150 mg/kg bw/day, reduced absolute and/or relative weights of the thymus, testes, prostate, epididymides and seminal vesicles were noted (see Table 4 under “Any other information on results incl. tables”). No toxicologically relevant findings were noted for the females. All other statistically significant changes were considered not to be a sign of toxicity as they occurred in the absence of a treatment-related distribution, were due to the lower terminal body weight or were considered due to the pregnancy/lactation status of the females (i.e. for the liver, adrenals, spleen, ovaries).

GROSS PATHOLOGY (PARENTAL ANIMALS)
Testes of reduced size (with or without flaccidity) were present in 8/10 males at 100 mg/kg bw/day and 8/10 males at 250/150 mg/kg bw/day. Epididymides of reduced size (with or without flaccidity) were present in 7/10 males at 100 mg/kg bw/day and 7/10 males at 250/150 mg/kg bw/day. Fore-stomachs (non-glandular portion) had irregular surfaces in 7/10 males and 5/10 females at 250/150 mg/kg bw/day. This observation was also noted in the glandular stomach of 3/10 males at 250/150 mg/kg bw/day. One male and one female at 100 mg/kg bw/day and two males at 250/150 mg/kg bw/day had reduced thymus size. The male animals that did not survive until planned necropsy showed additionally several abnormalities in the following organs: lungs, stomach, liver, seminal vesicles, spleen, mesenteric and mandibular lymph nodes, lacrimal glands, diaphragm, and body cavities. In addition, beginning autolysis was noted for the two animals that were found dead. Macroscopic examination of the female animal that was killed in extremis revealed findings in the uterus, cervix, spleen, mandibular lymph nodes, upper jaw, and abdominal cavity.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy observations included findings in the lungs (foci), liver (reduced in size), kidneys (pelvic dilation), epididymides (nodule), mandibular lymph nodes (enlarged, discolouration), uterus (enlarged, thickened, contains fluid), clitoral glands (discolouration), skin (alopecia), and spleen (nodule, enlarged).

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test article related microscopic observations were present in the stomach, testes, epididymides, spleen, and liver. Secondary effects were present in the uterus, female adrenals, female mammary glands, and thymus (see Table 5 under “Any other information on results incl. tables”).
Abnormal findings in the stomach included inflammation, haemorrhage, hyperkeratosis and hyperplasia of non-glandular epithelium, and degeneration of glandular epithelium. Incidences of pathological findings in the stomach are summarised in Table 5 under “Any other information on results incl. tables”.
In the testes, degeneration of seminiferous tubular epithelium, increased severity of edema, chronic active inflammation, and enlarged amphophilic cells were observed. Incidences of pathological findings in the testes are summarised in Table 5 under “Any other information on results incl. tables”. Staging of spermatogenesis in testes provided evidence of test article-related impairment to the spermatogenetic cycle at all dose levels in a dose related manner. The most subtle changes were those of enlarged spermatagonia with finely granular cytoplasm and asynchronous tubules in which normal cell associations were absent. Individual cell necrosis, spermatidic giant cells, and reduced layers of spermatagonia were common observations. At the 250/150 mg/kg bw/day dose level, there were no recognizable stages. Tubules were lined often by a single layer of cells with uncertain identity (Sertoli cells, primary spermatagonia or both). Mitotic figures were occasionally present and appeared abnormal.
Epididymal observations included degenerate sperm, hypospermia, atrophy and inflammation. Incidences of pathological findings in the epididymides are summarised in Table 5 under “Any other information on results incl. tables”.
In the spleen, reduced (but not dose-related) numbers of hematopoietic cells were noted.
Minimal or slight hepatocellular necrosis was observed in the liver of two males and one female at 250/150 mg/kg bw/day.
Secondary effects of treatment were observed in the uterus, adrenal gland, mammary gland and thymus. In the uterus, implantation sites were observed in 5/5, 6/6, 3/8 and 1/6 females at 0, 40, 100 and 250/150 mg/kg bw/day, respectively. Another three females of the 100 mg/kg bw/day group had placental trophoblasts and necrotic debris in the uterine lumen as evidence of litter loss. There was evidence of cyclic changes in the reproductive tracts of non-pregnant 250/150 mg/kg bw/day females. No pregnancy associated hypertrophy of the cortical adrenal was present in the females of all dose groups. In the mammary gland, observations consisted of reduced incidences and severities of normally expected pregnancy-related hyperplasia of the mammary gland. The incidences of hyperplasia were 5/5, 4/4, 4/5 and 0/5 in the respective 0, 40, 100 and 250/150 mg/kg bw/day dose groups with progressive decreases (3.0, 2.3, 1.5 and 0) in average severity. In the thymus, lymphoid cortical atrophy was present in 1/4 males at 100 mg/kg bw/day and 4/10 males 250/150 mg/kg bw/day. In females, these effects occurred in 1/5, 2/6 and 1/5 females at 40, 100 and 250 mg/kg bw/day, respectively. The unscheduled deaths at 100 and 250 mg/kg bw/day showed marked atrophy of the thymus. In the remaining animals, the incidence of the effect was only minimal or slight.

HAEMATOLOGY:
Several haematological parameters were statistically significantly affected by treatment with the test substance. Decreased levels of haemoglobin, mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC) and platelets were noted for males treated at 250/150 mg/kg bw/day. For females, decreased levels of haemoglobin, mean corpuscular volume (MCV), MCH, MCHC and increased prothrombin time were noted at 100 and 250/150 mg/kg bw/day. In addition, levels of MCV and MCH were also statistically significantly decreased for females treated at 40 mg/kg bw/day.

Effect levels (P0)

1

Key result

Dose descriptor:

LOAEL

Remarks:

fertility

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: females: increase in precoital time, increased duration of pregnancy; other: histopathological alterations in testes and epididymides

2

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: females: levels of MCV and MCH were statistically significantly decreased; males: histopathological alterations in the testes and epididymides

3

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

< 40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: females: increase in precoital time, increased duration of pregnancy; males: histopathological alterations in testes and epididymides

4

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

< 40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: females: levels of MCV and MCH were statistically significantly decreased; males: histopathological alterations in the testes and epididymides

5

Dose descriptor:

NOAEL

Remarks:

developmental

Effect level:

< 40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: viability index; postnatal loss

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

40 mg/kg bw/day: lean and pale appearance

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

40 mg/kg bw/day: 3/10 litters dead, only 4 litters with some live pubs survived; 100 and 250/150 mg/kg bw/day: no litters

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

40 mg/kg bw/day: slightly decreased body weights

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):

40 mg/kg bw/day: autolysis in dead animals, blue discolouration of the snout, absence of milk in stomach

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING): see Table 6 under "Any other information on results incl. tables"
A clear dose-related effect on development was noted at 40, 100 and 250/150 mg/kg bw/day. Implantation sites were only noted for nine females at 100 mg/kg bw/day and two females at 250/150 mg/kg bw/day. The remaining females were non pregnant or did not mate. Therefore, no pups could be examined for postnatal development. Out of the nine litters at 40 mg/kg bw/day, only six had live pups at first litter check. The number of pups per litter was decreased when compared to the control group. In addition, most of these pups did not survive the first days of lactation; only four litters had some live pups at planned necropsy.

CLINICAL SIGNS (OFFSPRING)
At 40 mg/kg bw/day, lean and pale appearance was seen in the surviving pubs.

BODY WEIGHT (OFFSPRING)
Body weights of the pups at 40 mg/kg bw/day were slightly, but not statistically significantly decreased when compared to the control group.

GROSS PATHOLOGY (OFFSPRING)
Macroscopic findings involved absence of milk in the stomach and blue discolouration of the snout. In addition, autolysis was noted for pups found dead.

Effect levels (F1)

Key result

Dose descriptor:

LOAEL

Remarks:

developmental

Generation:

F1

Effect level:

40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: viability index; postnatal loss

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 3. Reproduction data

Parameter	Dose [mg/kg bw/day]
	0	40	100	250/150
Mating index [%]	100	100	100	90
No. of females mated	10/10	10/10	10/10	9/10
Fertility index [%]	100	100	90	20
No. of implantation sites#	11.1 ± 2.0	9.8 ± 1.4	7.8 ± 4.3	3.5 ± 3.5
No. of corpea lutea#	14.5 ± 4.3	11.3 ± 1.8	9.9 ± 4.5	1.1 ± 3.0**
Duration of gestation [d]#	21.4 ± 0.5	23.1 ± 0.6	n.a.	n.a.
Conception index [%]	100	100	90	22.2
No. of pregnant females	10/10	10/10	9/10	2/10
No. of non-pregnant females	0/10	0/10	1/10	8/10
No. of females with live pups (Day 1)	10/10	7/10	0/10	0/10
Gestation index [%]	100	70	0	0
Litter size	10	9	n.a.	n.a.

*/** Steel−test significant at 5% (*) or 1% (**) level, n.a. = not applicable; # mean value ± standard deviation

Table 4. Statistically significant changes in reproduction organ weights of males

Organ weights	Dose [mg/kg bw/day]
	0	40	100	250/150
Thymus, absolute [g]	0.260 ± 0.017	0.258 ± 0.033	0.141 ± 0.014**	0.116 ± 0.022**
Thymus, relative [%]	0.079 ± 0.006	0.083 ± 0.011	0.048 ± 0.006**	0.040 ± 0.007**
Testis, absolute [g]	3.31 ± 0.18	3.26 ± 0.31	1.86 ± 0.30**	1.46 ± 0.11**
Testis, relative [%]	1.02 ± 0.12	1.03 ± 0.07	0.62 ± 0.10**	0.51 ± 0.05**
Epididymides, absolute [g]	1.133 ± 0.106	1.104 ± 0.077	0.801 ± 0.091**	0.645 ± 0.068**
Epididymides, relative [%]	0.348 ± 0.043	0.349 ± 0.027	0.268 ± 0.040**	0.225 ± 0.027**
Seminal vesicles, absolute [g]	1.778 ± 0.222	1.460 ± 0.138	1.377 ± 0.201*	1.420 ± 0.213*
Seminal vesicles, relative [%]	0.543 ± 0.067	0.468 ± 0.049	0.471 ± 0.065	0.482 ± 0.065
Prostate, absolute [g]	0.662 ± 0.138	0.549 ± 0.111	0.471 ± 0.075*	0.413 ± 0.042*
Prostate, relative [%]	0.201 ± 0.038	0.176 ± 0.035	0.161 ± 0.025	0.140 ± 0.016*

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 5. Histopathological findings in reproduction organs/endocrine organs of males and females

Histopathological findings in reproduction organs/endocrine organs	Dose [mg/kg bw/day]
	0	40	100	250/150
Number of animals per group	10	10	10	10
Primary effects
TESTES (males)	5	5	9	10
- Enlarged cells	-	-	-	2
- Degeneration of seminiferous tubular epithelium	1	2	9	8
- Edema	4	3	7	7
- Chronic active inflammation	-	-	-	2
- dilated rete	-	1	1	-
TESTES, PAS STAGING (males)	5	5	5	8
- All stages missing	-	-	1	5
- Enlarged cells	-	-	-	2
- Most stages missing	-	-	4	1
- Some stages missing	-	-	-	1
- Multiple acrosomes	-	-	-	1
- Asynchronous tubules	-	2	4	1
- Individual cell necrosis	-	3	3	-
- Reduced spermatagonia	-	1	5	6
- Spermatidic giant cells	-	1	3	1
- Vacuolation basilar	-	-	1	-
EPIDIDYMIDES (males)	5	5	8	10
- Sperm granuloma	-	1	1	-
- Sperm degeneration	-	-	8	8
- Hypospermia	-	-	1	8
- Atrophy	-	-	7	8
- Chronic active inflammation	-	-	1	4
Secondary effects
UTERUS (female)	5	6	8	6
- Stromal hyperplasia	-	-	-	1
- Dilation cyclic	-	-	-	3
- Haemorrhage	2	6	3	-
- Inflammation supp	-	-	1	-
- Necrotic debris/neut	-	-	1	-
- Implant sites	5	6	3	1
- Throphoblasts/Necro	-	-	3	-
ADRENALS(females)	5	-	1	5
- Hypertrophy cortex	5	-	-	-
- Extra cortical nodule	1	-	-	-
- Extramed haematopoies	-	-	1	-
MAMMARY GLAND AREA(females)	5	4	5	5
- Hyperplasia	5	4	4	-
- Inactive gland	-	-	1	5
- Active gland	-	1	1	-
- Infiltrate lymphoid	-	1	-	-
THYMUS (females)	5	5	6	5
- Increased apoptosis	-	1	-	-
- Haemorrhage/Congestion	-	1	1	-
- Athrophy lymphoid	-	1	2	1
- Hyperplasia duct	-	1	-	-
THYMUS (males)	5	5	4	8
- Increased apoptosis	-	1	2	-
- Haemorrhage/Congestion	-	1	1	1
- Athrophy lymphoid	-	-	1	4

Table 6. Developmental data

Parameter	Dose [mg/kg bw/day]
	0	40	100	250/150
Pub weight [g]#	6.0 ± 0.7	5.6 ± 0.5	n.a.	n.a.
Sex ratio [% males]	42	43	n.a.	n.a.
Viability index [%]	99	66.7**	n.a.	n.a.
Litter size	10	9	n.a.	n.a.
Dead pubs at first litter check
- Litters affected	0/10	6/10**	n.a.	n.a.
- Total	0	13	n.a.	n.a.
Living pups at first litter check	103	30	n.a.	n.a.
- Mean per litter	10.3 ± 2.4	3.3 ± 3.2++	n.a.	n.a.
Postnatal loss
- Litters affected	1/10	1/9	n.a.	n.a.
- Total	1	10**	n.a.	n.a.
- % of living pups	1.0	33	n.a.	n.a.

*/** Fisher's Exact test significant at 5% (*) or 1% (**) level; +/++ Steel−test significant at 5% (+) or 1% (++) level; n.a. = not applicable; # mean values ± standard deviation

Applicant's summary and conclusion

Conclusions:

CLP: Repr. Cat. 1B, H360FD