Octahydro-2H-1-benzopyran-2-one

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Fertility (OECD TG 422): NOAEL = 386 mg/kg bw/day in females after dietary exposure of 6000 ppm. In males no effects were found at >=452 mg/kg bw(after dietary exposure of 6000 ppm).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August 2016 - 19 october 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

july 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

july 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/D evelopmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28- day oral toxicity study in rodents

Version / remarks:

July 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

October 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

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

Details on species / strain selection:

This species and strain of rat has been recognized as appropriate for general and reproduction toxic ity studies. Charles River Den Bosch 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.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: approximately 10 weeks; Females: approximately 12 weeks.
- Weight at study initiation: males 297-334 g; females 197-236 g
- Fasting period before study: no
- Housing: pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages; premating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages; mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages; post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages; lactation: Pups were kept with the dam until termination in Macrolon plastic cages. 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.
general: Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: Free access to prepared diets. During the acclimatization period, animals had free access to
standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours. The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage. The same diets remained in the food hopper for a maximum of 1 day. On the day of weighing the remaining food in the food hopper, remaining diet was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.
- Water: Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 August 2016 To: 19 October 2016

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C, for a maximum of 16 days) until use, if not used on the day of preparation.

Details on mating procedure:

Details on mating procedure
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 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 (how): females were housed individually with nesting materials

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase according to a validated method.
Group Analysis (type of sample)
1 acc (M)
2 acc + hom (TMB)
3 acc (M)
4 acc + hom (TMB)
Duplicate samples were analyzed
Type of sample T=Top, M=Middle, B=Bottom position of container
Analysis acc=accuracy, hom=homogeneity
The accuracy of diet preparations was considered acceptable if the mean measured concentrations was 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of diet preparations at 500 and 15,000 ppm was determined over 18 days at room temperature and over 16 days in the freezer (≤-15°C) prior to conduct of the dose range finding study, as part of the analytical method development and validation study. Based on these stability results over 8 days at room temperature, additional stability analyses were conducted over 1, 2 and 4 days at room temperature prior to conduct of the dose range finding study.
In addition, random back-up diet samples were taken and stored at ≤-15ºC for possible future analysis. Any remaining samples at finalization of the study report were discarded. Sampling occurred as soon as possible after preparing the diets. If the analytical determinations were not performed on the day of preparation, the samples were stored at ≤-15ºC.

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were exposed for 50 - 57 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were exposed for 41 or 43 days.
Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces or spilled diet from the food hopper.

Frequency of treatment:

ad libitum

Dose / conc.:

1 000 ppm

Remarks:

equivalent to 66 mg/kg bw

Dose / conc.:

3 000 ppm

Remarks:

equivalent to 200 mg/kg bw

Dose / conc.:

6 000 ppm

Remarks:

equivalent to 400 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Doses for the main study were selected based on the results of a dose range finder study. In this study 5 males and 5 females were administered at dietary dose levels of 0, 5000 (equivalent to 333 mg/kg bw) and 15,000 ppm (equivalent to 1000 mg/kg bw) for 14 days.
At 15,000 ppm, hunched posture was recorded for 2/5 females, reduced bodyweight gain of
males and females (10-15%) and reduced food consumption (both sexes) were noted. The
relative liver and kidney weights of males were significantly increased and an extended
diestrus for 4/5 females were seen. Histopathology at 15,000 ppm showed possible test item related moderate bilateral tubular degeneration in the testes, with cell debris and reduced luminal sperm in the epididymides in 1/5 males, and mucification of the vagina (up to moderate degree) with cellular debris in 4/5 females.
At 5000 ppm, reduced bodyweight gain and mainly in females reduced food consumption were noted on some days, also increased liver weights of males and females were noted.
Based on these results, dose levels of 1000, 3000 and 6000 ppm were selected for the main study.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: 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 PND 1, 4, 7 and 13.

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n)
- How many animals: all
- Parameters checked according to Guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes (o/n)
- How many animals: all
- Parameters checked according to Guideline

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: males during week 4 of treatment, females during the last week of
lactation (e.g. PND 6-13)
- Dose groups that were examined: 5/sex/dose
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore-and hind-limb strength, locomotor activity.

IMMUNOLOGY: No

OTHER: estrous cycle determination and general reproduction data.
Determination plasma T4 and TSH levels at necropsy

Oestrous cyclicity (parental animals):

yes (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy)

Sperm parameters (parental animals):

Parameters examined in F1 males:
testis weight, epididymis weight., histopatology to examine staging of spermatogenesis from all males of Groups 1 and 4 and all males of Groups 1 and 4 and all males that failed to sire.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter of equal sex distribution (if possible); Blood samples were collected from two of the surplus pups for determination of Thyroid hormone level.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), presence of nipples/areolae in male pups, determination blood T4 and TSH levels (PND 4 pups and PND 13-15 pups)

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

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes according to guideline.
F1: All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development. At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling, was preserved in 10% buffered formalin.

HISTOPATHOLOGY: Yes according to guideline

Other examinations
Organ weights from 5 selected animals/sex/group: Adrenal glands, Brain, Cowper’s glands,Epi
didymides, Glans penis, Heart, Kidneys, Levator ani plus bulbocavernosus muscle complex (LABC),
Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid (including parathyroid if detectable), Uterus (including cervix).
Organ weights from all remaining animals: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid.

Examinations of pups (see section 7.8.2)

Postmortem examinations (offspring):

SACRIFICE
Recognizable fetuses of the female killed in extremis was examined externally, sexed (if possible), and euthanized by decapitation. Pups, younger than 7 days were killed by decapitation.
All remaining pups (PND 7-15) were sacrificed using Euthasol® 20% by intraperitoneal (ip) injection.
On PND 4 (at culling), from 2 pups per litter, blood samples (0.4 mL in total) were collected by decapitation. Blood samples from the 2 pups per litter were collected into one serum tube.
On PND 13-15, from 2 pups per litter (if possible from one male and one female13) blood samples were collected at planned necropsy. As part of the necropsy procedure, blood samples (0.9 mL) were drawn by aorta puncture under anaesthesia using isoflurane, followed by exsanguination. Blood was collected into serum tubes.

GROSS NECROPSY
All pups were sexed by both external as well as internal examination. Descriptions of all abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
At terminal sacrifice (PND 13-15), the thyroid from 2 pups per litter, i.e. the same pups as selected for blood sampling (see also section 5.11 and 5.13.1), was preserved in 10% buffered formalin.

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 ttest) 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.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motoractivity data to determine intergroup differences.
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.

Reproductive indices:

For each group, the following calculations were performed:
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 pups/ Number of pregnant females) x 100

Duration of gestation = Number of days between confirmation of mating and the beginning of parturition

Offspring viability indices:

Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100

Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.

Live birth index (%) = (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100

Percentage live males at First Litter Check (%) = (Number of live male pups at First Litter Check/ Total 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/ Total number of live pups at First Litter Check) x 100

Viability index (%) = (Number of live offspring on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100

Lactation index (%) = (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100

Group mean values were calculated from individual litter values.

Sex ratio (percentage males) = (Number of males in litter/ Total number of offspring in litter) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or duringweekly arena observations.
Any clinical signs noted among surviving animals during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent doserelated trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 1000 ppm was sacrificed moribund on Day 4 of the lactation period following an accidental injury. Following temporary unconsciousness, this animal displayed signs of ill health and deficiency in maternal care. Histopathologically, the cause of moribundity for this animal was considered to be mucosal necrosis in the jejunum, which was supported at necropsy by dark red focus/foci on the jejunum and blood/blood clots in the abdominal cavity.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes in body weights or body weight gain were noted in males up to and including 3000 ppm and in females at 1000 ppm.
At 6000 ppm, body weights and body weight gain of males and females were lower than controls throughout treatment, achieving a level of statistical significance on most occasions. At the end of treatment, the difference in absolute mean weight to controls was approximately 7% for males. For females, the difference in absolute mean weight to controls was approximately 9% at the end of post-coitum and approximately 16% at the end of lactation. For a few of these females, slight weight loss (ranging from 3 to 8%) was recorded during lactation, and during the first week of the treatment period.
At 3000 ppm, body weights and body weight gain of females were lower than controls during the lactation period only, achieving a level of statistical significance on most occasions. Slight weight loss (2 or 3%) was recorded for a few of these females during lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 6000 ppm, a lower mean food consumption before and after allowance for body weight was recorded for females during the post-coitum and most predominantly during the lactation period. The overall mean food intake during lactation was approximately 37% lower than controls.
At 3000 ppm, mean food consumption of females was lower in the second week of the lactation period, but differences from control values were not statistically significant.
No treatment-related changes in food consumption before or after allowance for body weight were noted in males up to 6000 ppm and in females at 1000 ppm.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in haematology parameters distinguished treated from control animals:
• Elongated prothrombin time (PT) in males and females at 6000 ppm.
• Higher red blood cell counts in females at 6000 ppm.
• Lower reticulocyte counts in males and females at 6000 ppm (not statistically significant for males).
• Lower haematocrit in males at 3000 and 6000 ppm.
• Lower mean corpuscular volume (MCV) in females at 6000 ppm.
• Lower mean corpuscular haemoglobin (MCH) in females at 6000 ppm.
No treatment-related changes in haematology parameters were noted in males and females treated at 1000 ppm. Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
• Higher alanine aminotransferase activity (ALAT) in males at 6000 ppm.
• Higher aspartate aminotransferase activity (ASAT) in males at 6000 ppm.
• Lower total protein in males at 6000 ppm, and in females at 3000 and 6000 ppm.
• Lower albumin in males at 6000 ppm, and in females at 3000 and 6000 ppm.
• Higher total bilirubin in females at 3000 and 6000 ppm.
• Higher urea in males at 6000 ppm.
• Lower creatinine in males at 6000 ppm.
• Lower cholesterol in females at 6000 ppm.
• Higher bile acids in males at 6000 ppm and in females at 3000 and 6000 ppm (not statistically significant for females; higher mean at 6000 ppm was mainly due to one female.
• Higher potassium in males at 6000 ppm.
• Lower calcium in males at 6000 ppm.
No treatment-related changes in clinical biochemistry parameters were noted in males treated up to and including 3000 ppm and in females treated at 1000 ppm. The apparent higher mean alkaline phosphatase activity (ALP) in females at 3000 and 6000 ppm was due to a high interindividual variation which could not be attributed to treatment-related histopathological changes. As such these were considered to have occurred by change and to be of no toxicological significance. Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and treated animals.
Motor activity data showed a similar habituation profile with a decreasing trend in activity over the duration of the test period for all groups.
For males treated at 6000 ppm an apparent trend towards lower mean values for total movements and ambulations was recorded. This was not considered toxicologically relevant since these variations were not statistically significant and remained within the range considered normal for rats of this age and strain, and habituation profile of these animals was similar to that encountered for control animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were present in the liver of males and females treated at 3000 and 6000 ppm, in the spleen of males and females treated at 6000 ppm, in the thymus of females treated at 3000 and 6000 ppm, and in the thyroid gland of males treated at 6000 ppm, and are summarized in text tables A, B and C (see "other information").
The following changes were observed in the liver:
• Inflammatory cell infiltrate peribiliary was present at increased incidence and severity in males and females treated at 3000 and 6000 ppm up to moderate degree.
• Single cell necrosis peribiliary was present in females treated at 3000 and 6000 ppm up to moderate degree and in males treated at 6000 ppm at minimal degree.
• Hepatocellular hypertrophy was present in females treated at 6000 ppm at slight degree.
In the spleen, a decreased incidence and severity of extramedullary hematopoiesis was present in males and females treated at 6000 ppm. For females this correlated with the decreased spleen weights at 6000 ppm.
In the thymus, lymphoid atrophy was present in females treated at 3000 and 6000 ppm up to slight degree. For a single female at 6000 ppm this correlated with the macroscopic finding reduced in size. This correlated with the lower thymus weights in females at 6000 ppm.
In the thyroid gland, follicular cell hypertrophy was present at increased incidence and severity in males treated at 6000 ppm up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Serum T4 levels of F0 males were not considered to be affected by treatment. The statistically significantly higher T4 value in males treated at 3000 ppm was not considered treatment-related since a dose-related response was absent and the mean remained within the range considered normal for rats of this age and strain.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Length and regularity of the estrous cycle were not affected by treatment.
Most females had regular cycles of 4 days. Two females at 6000 ppm had an irregular cycle during the treatment period. One of these females had healthy offspring, the other female had a normal number of implantation sites but failed to deliver. This incidence of females with irregular cycles was within normal limits and therefore not considered to be related to treatment.

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Description (incidence and severity):

Mating index was not affected by treatment. All females showed evidence of mating. Fertility and conception index were not affected by treatment. One female at 1000 ppm and one female at 6000 ppm were not pregnant (as confirmed by Salewski staining). In the absence of a dose-related incidence, this non-pregnancy was not considered to be related to treatment.
Precoital time was not affected by treatment.
At 6000 ppm, the mean number of implantation sites was statistically significantly lower compared to controls (9.9, versus 11.9 in the control group).

At 6000 ppm, a lower number of implantation sites was recorded.
No treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). In males, no reproductive toxicity was seen in the reproductive organs.

Key result

Dose descriptor:

NOAEL

Effect level:

386 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: reduced number of implantation sites at 6000 ppm

Remarks on result:

other: dietary concentration was 3000 ppm

Key result

Dose descriptor:

NOAEL

Remarks:

female

Effect level:

128 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: dietrary concentration was 1000 ppm

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs were noted in all pups of three females treated at 6000 ppm (8, 8 and 1 surviving pups, respectively). A (very) lean
appearance was noted for the surviving pups of 2 females at PND 13 and/or 14, and in the pup of one female from PND 4 onwards. The lean appearance correlated with the lower pup body weights at 6000 ppm. Additionally, the pups of these females were dehydrated at PND 13 and 14, and the pup with lean appearance from PND 4 onwards had little milk in the stomach at PND 7, 8 and 10.
No other clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of the clinical signs observed incidentally at 1000 and 3000 ppm remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices in the different groups ranged from 98 to 100%.
One control female and one female at 6000 ppm had a dead pup at first litter check. This incidental pup mortality was unrelated to treatment with the test item.
The viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices in the different groups ranged from 90% (1000 ppm) to 98% (3000 and 6000 ppm) or 100% (control group).
Post-natal loss up to PND 4 was limited to one pup at 1000 ppm which went missing on PND 2, two pups at 3000 ppm that went missing on PND 3 or 4, and one pup at 6000 ppm which went missing on PND 2. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The statistically significantly higher total number of pups lost between PND 1-4 at 1000 ppm can be explained by the premature sacrifice of one female together with her eight healthy pups. The moribund condition of this female was not related to treatment (see results F0 generation).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 3000 and 6000 ppm, lower mean body weights were recorded for male and female pups from PND 1 onwards. At PND 1, mean body weights were decreased by about 5% (3000 ppm) or 10% (6000 ppm), and at PND 13 these differences had increased to 13% (3000 ppm) and 39% (6000 ppm). The differences were statistically significant on most occasions in the 6000 ppm group and at PND 13 in the 3000 ppm group.
Pup body weights at 1000 ppm were not considered affected by treatment.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 6000 ppm, all pups of two litters had a lean appearance. A lean appearance was also noted up to the day prior to scheduled sacrifice in the single pup of one female from dose group 6000 ppm and most likely this pup was lean at sacrifice (inadvertently, macroscopic findings of this pup were not recorded). Additionally, all pups of one of the litters with lean appearance had a gastrointestinal tract distended with gas. These findings correlated with the lower pup body weights at 6000 ppm and were considered to be related to the treatment.
No other macroscopic findings were noted among pups that were considered to be related to treatment. The incidental macroscopic findings noted in the two pups found dead at first litter check (one pup at 0 and one pup at 6000 ppm) were unrelated to treatment as they represented normal background findings and occurred incidentally. No macroscopic findings were noted in the pups of the female from dose group1000 ppm which was sacrificed moribund during lactation.

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was not affected by treatment.
Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Treatment up to and including 6000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.

Developmental data:
Gestation index and duration of gestation were not affected by treatment. A total of two females each at 1000 and 6000 ppm had no offspring, resulting in a gestation index of 89% at both these dose levels. One female each at 1000 and 6000 ppm had implantation sites only, and one female each at 1000 and 6000 ppm were not pregnant. The incidence of absence of offspring at these dose levels showed no relationship to the dose and was considered to be within the range considered normal for rats of this age and strain. As such, this was not considered to be related to treatment.
No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed among the surviving animals.
The lactation index (number of live offspring on PND 13 as percentage of number of live offspring on PND 4 after culling) was not affected by treatment. The lactation indices in the different groups were 99% (3000 ppm) or 100% (0, 1000 and 6000 ppm). The incidental loss of one pup at 3000 ppm that went missing on PND 10 was not considered related to treatment since no dose-related trend in incidence was recorded.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

The reproduction and developmental toxicity observed in this study occurred at dose levels associated with parental toxicity. This included lower mean body weights of 16% for females, accompanied by a nearly 40% lower food consumption during the lactation period at 6000 ppm. It could not be excluded that this effect on body weight and food intake was related to the observed reproduction and developmental toxicity. No reproduction or developmental toxicity was observed at dose levels which were non-toxic to the parents.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

386 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

other: lower litter size and post-implantation survival

Remarks on result:

other: Dietary concentration was 3000 ppm

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

other: lower body weights and body weight gain of pups

Organ:

other: body weight (gain)

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

yes

The concentrations analyzed in the diets of Groups 2 - 4 (1000, 3000 and 6000 ppm, respectively) were in agreement with target concentrations with mean accuracies between 106 and 109).

No test item was detected in the Group 1 diet.

The diets of Group 2 (1000 ppm) and Group 4 (6000 ppm) were homogeneous, with coefficient of variation ranging from 2.4 to 2.8.

Analysis of the diets at 500 ppm (low) and 15000 ppm (high) after storage yielded a relative differences of ≤ 10% (actual data -3.1% to -8.5%) when stored for 1 or 2 days and > 10% (actual data -19% and -16% in low and high dose groups, espectively) when stored for 4 days. Based on this, the diets were considered to be stable during storage at room temperature under normal laboratory light conditions

for 2 days.

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table below (mg/test item/kg bw/day, overall group means in the indicated period)).

Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((14 x mean premating) + (12 x mean mating)) / 26

Females: ((14 x mean premating) + (21 x mean post-coitum) + (13 x mean lactation)) / 48

	Group 2 1000 ppm	Group 3  3000 ppm	Group 4  6000 ppm
Males
Pre-mating	75	226	466
Mating	67	203	435
Mean of means	71	215	452
Females
Pre-mating	73	226	416
Post-coitum	109	324	592
Lactation	218	659	950
Mean of means	128	386	638

Organ weights

Mean Percent Liver, Thymus, Spleen and Kidneys Weight Differences from Control Group ( *: P<0.05, **: P<0.01)

	Males			Females
Dose level (ppm)	1000	3000	6000	1000	3000	6000
Liver absolute	0	9	2	-1	13	-16
Liver relative to BW	-1	8	13**	1	21	1
Thymus absolute	13	-1	-26	-1	-16	-30*
Thymus relative to BW	12	-2	-18	-4	-9	-17
Spleen absolute	18	11	-3	-3	-10	-22*
Spleen relative to BW	17	10	7	-1	-2	-6
Kidneys absolute	-2	11	4	-3	-1	-8
Kidneys relative to BW	-3	9	15*	-3	7	11

Microscopic examination

Table A Summary Test Item-Related Microscopic Findings – Spleen and Liver – Both Sexes

(a )= Number of tissues examined from each group.

	Males				Females
Dose level (ppm)	0	1000	3000	6000	0	1000	3000	6000
Liver	5	5	5	5	5	5	5	5
Inflammatory cell infiltrate peribiliary
Minimal	1	2	4	-	-	-	3	-
Slight	-	-	-	3	-	-	2	4
Moderate	-	-	-	2	-	-	-	1
Single cell necrosis peribiliary
Minimal	-	-	-	2	-	-	3	2
Slight	-	-	-	-	-	-	-	3
Moderate	-	-	-	-	-	-	1	-
Hepatocellular hypertrophy
Slight	-	-	-	-	-	-	-	2
Spleen	5	5	5	5	5	5	5	5
Hematopoiesis, extramedullary
Minimal	3	4	4	-	4	4	2	1
Slight	1	-	1	-	1	-	2	-

Table B.

Summary Test Item-Related Microscopic Findings – Thymus - Females Only

(a) = Number of tissues examined from each group.

	Females
Dose level (ppm)	0	1000	3000	6000
Thymus (a)	5	5	5	5
Atrophy lymphoid
Minimal	-	-	2	3
Slight	-	-	-	1

Table C

Summary Test Item-Related Microscopic Findings – Thyroid Gland - Males Only

(a) = Number of tissues examined from each group

	Males
Dose level (ppm)	0	1000	3000	6000
Thyroid gland (a)	5	5	5	5
Hypertrophy follicular cell
Minimal	2	3	3	2
Slight	-	-	-	2

Conclusions:

In conclusion, treatment with the substance dosed via diet at dose levels 1000, 3000 and 6000 ppm resulted in a NOAEL for parental toxicity of 1000 ppm (corresponding to a mean test item intake of 128 mg/kg bw/day in females) based on histopathological changes in the liver characterized by the combination of peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis at 3000 ppm in females and 6000 ppm in males and females. The NOAEL for reproduction toxicity in this study is 3000 ppm (corresponding to a mean daily test item intake of 215 mg/kg bw/day in males and 386 mg/kg bw/day in females), based on a reduced number of implantation sites at 6000 ppm.

Executive summary:

In the executive summary on fertility the repeated dose toxicity section is repeated to present the context of the fertility effects. The developmental toxicity is presented in the specific record.

For Bicyclononalactone a combined repeated dose toxicity study with the reproductive/developmental screening test according to OECD 422 was performed with rat. Based on a range finding test the following concentrations in the diet were used, with dose groups 0, 1000, 3000 and 6000 ppm, aiming at doses of 66, 200 and 400 mg/kg bw. The following repeated dose toxicity parameters were recorded; mortality, clinical signs, functional observations and locomotor activity, body weight and food consumption, haematology parameters and organ weights, and macroscopy and histopathology on a selection of tissues. In addition thyroid hormone T4 (F0 males at the end of treatment and PND 13 -15 pups) were measured. The parameters assessed for fertility and developmental toxicity are described at the respective sections. The doses were confirmed by analysis.

Analysis of the test substance intakeshowed that the dietary concentrations of 1000, 3000 and 6000 ppms resulted in doses for males: 71, 215 and 452 mg/kg bw and for females 128, 386, 638 mg/kg bw, respectively. 

Clinical signs and FOB: There were no treatment-related changes in clinical appearance and functional observations.

Body weight and food consumption: Lower body weight and food consumption was recorded at 3000 and 6000 ppm. At 6000 ppm, body weight and body weight gain was lower in males and females throughout the treatment period with occasional slight weight loss for females during the first week of treatment and during lactation. At the end of treatment, mean body weight was approximately 7 and 16% lower than controls for males and females, respectively. For females, this was accompanied by a lower food consumption during the post-coitum period and, more markedly (nearly 40% difference from controls), during the lactation period. Since lower body weight gain was also noted in males, a direct test item related effect was considered likely. At 3000 ppm, body weight and body weight gain was lower for females during the lactation period, along with lower food intake in the second week of the lactation period. Despite the lower body weights and food intake at 3000 and 6000 ppm, animals appeared in a good health condition based on absence of treatment-related changes in other in-life parameters including mortality, clinical appearance and functional observations. There were no obvious treatment-related changes in other in-life parameters including mortality, clinical appearance and functional observations. At 1000 ppm, body weight and food consumption were considered unaffected by treatment.

Haematology: Some treatment related but non-adverse effects were noted at 6000 ppm. Minor haematological changes were noted in males and/or females primarily consisting of lower reticulocyte counts (approximately 40% lower than controls), and less pronounced changes consisting of higher red blood cell counts, and haematocrit, mean corpuscular volume and mean corpuscular haemoglobin. These changes were indicative on an effect on red blood cell turn over, and could be related to the morphological changes in the spleen (decreased incidence and severity of extramedullary haematopoiesis, and lower spleen weights in females). Marginally lower haematocrit recorded for males at 3000 ppm had no morphological correlates and at 1000 ppm, haematological parameters were considered unaffected by treatment.

Clinical biochemistry: Some non-adverse effects were seen. Increases in alanine and aspartate aminotransferase activity, total bilirubin and bile acids in males and/or females at 6000 ppm could be related to the histopathological liver effects (see the discussion at that section). Other clinical biochemistry changes that were considered to be treatment-related consisted of decreases in total protein and albumin in males at 6000 ppm and females at 3000 and 6000 ppm, higher potassium and urea and lower creatinine and calcium in males at 6000 ppm, and lower cholesterol in females at 6000 ppm. These changes were generally mild in degree and essentially remained within the range considered normal for rats of tis age and strain. Additionally, since these variations had no apparent treatment-related morphological correlates, these were not considered adverse.

Liver effects: At 3000 ppm (females) and 6000 ppm (both sexes), adverse, treatment-related changes in the liver were recorded. These consisted of a combination of peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis. Increases in alanine and aspartate aminotransferase activity, total bilirubin and bile acids in males and/or females at 6000 ppm could be related to the peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis noted in the liver of these males. An increased incidence of peribiliary inflammatory cell infiltrate was also noted in males at 3000 ppm. Based on its minimal severity and the absence of any degenerative changes such as single cell necrosis, this finding in 3000 ppm treated males was considered to be non-adverse. Additionally, two females at 6000 ppm showed slight hepatocellular hypertrophy.

Spleen effects: Non-adverse effects were seen. At 6000 ppm, a decreased incidence and severity of extramedullary haematopoiesis was recorded in the spleen of males and females at 6000 ppm. This correlated with lower spleen weights in females. A few minor haematological changes were noted at 6000 ppm in males and/or females that could be related to these morphological changes in the spleen, and consisted of higher red blood cell counts, and lower reticulocyte counts, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin. None of these haematological changes were however considered adverse, and therefore the morphological changes in the spleen were also not considered adverse.

Thymus effects: Non-adverse effects were seen. At 3000 and 6000 ppm, minimal to slight lymphoid atrophy of the thymus was recorded for females. This correlated with lower thymus weights of females at 3000 and 6000 ppm and with a reduced thymus size in a single female of this group. This lymphoid atrophy was not accompanied by any other indicator of toxicity and was, at this degree, considered non-adverse.

Thyroid effects: Non-adverse effects were seen. At 6000 ppm, a minor increase in follicular cell hypertrophy (up to slight degree) in the thyroid of males was recorded. This was regarded to be an adaptive change and considered to be non-adverse at the incidences and severities recorded. No concurrent treatment-related change in thyroid weight was recorded.

Other organs: No adverse morphological changes were noted in any of the other organs examined in this study.

In conclusion for repeated dose toxicity, the key adverse changes were recorded in the liver at 3000 (females) and 6000 ppm (males and females) characterized by peribiliary inflammation/necrosis. Though a number of other changes are seen and may be treatment related and in view of the mild character were not considered adverse. These effects are covered with a NOAEL set at the low dose for females and the mid dose for males: 1000 and 3000 ppm, respectively. These values corresponds to a mean substance intake of 128 mg/kg bw for females and 215 mg/kg for males. The overall parental NOAEL is therefore 128 mg/kg bw.

 

Fertility effects:

No adverse effects were seen in males up to 6000 ppm relating to 452 mg/kg bw. Adverse effects were seen in females at a dose where also repeated dose effects were recorded. At 6000 ppm (nominally 400 mg/kg bw for dams), a lower number of implantation sites was recorded versus control 9.9 versus 11.9, without dose relation. No treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). The decreased implantation sites can be associated with lower mean body weights of females of 9% at the end of the post-coitum phase.

In conclusion for fertility: No adverse on male fertility was seen. The female LOAEL is 6000 ppm converted being 638 mg/kg bw. In the presence of a repeated dose toxicity of 128 mg/kg bw, the overall NOAEL for fertility is set at 386 mg/kg bw. 

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

386 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

For reproduction toxicity there is one oral study available, conducted according to OECD TG 422 in compliance with GLP, resulting in adequate information for this endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

For Bicyclononalactone a combined repeated dose toxicity study with the reproductive/developmental screening test according to OECD 422 was performed with rat. Based on a range finding test the following concentrations in the diet were used, with dose groups 0, 1000, 3000 and 6000 ppm, aiming at doses of 66, 200 and 400 mg/kg bw. The following repeated dose toxicity parameters were recorded; mortality, clinical signs, functional observations and locomotor activity, body weight and food consumption, haematology parameters and organ weights, and macroscopy and histopathology on a selection of tissues. In addition thyroid hormone T4 (F0 males at the end of treatment and PND 13 -15 pups) were measured. The parameters assessed for fertility and developmental toxicity are described at the respective sections. The doses were confirmed by analysis.

Analysis of the test substance intakeshowed that the dietary concentrations of 1000, 3000 and 6000 ppms resulted in doses for males: 71, 215 and 452 mg/kg bw and for females 128, 386, 638 mg/kg bw, respectively. 

Clinical signs and FOB: There were no treatment-related changes in clinical appearance and functional observations.

Body weight and food consumption: Lower body weight and food consumption was recorded at 3000 and 6000 ppm. At 6000 ppm, body weight and body weight gain was lower in males and females throughout the treatment period with occasional slight weight loss for females during the first week of treatment and during lactation. At the end of treatment, mean body weight was approximately 7 and 16% lower than controls for males and females, respectively. For females, this was accompanied by a lower food consumption during the post-coitum period and, more markedly (nearly 40% difference from controls), during the lactation period. Since lower body weight gain was also noted in males, a direct test item related effect was considered likely. At 3000 ppm, body weight and body weight gain was lower for females during the lactation period, along with lower food intake in the second week of the lactation period. Despite the lower body weights and food intake at 3000 and 6000 ppm, animals appeared in a good health condition based on absence of treatment-related changes in other in-life parameters including mortality, clinical appearance and functional observations. There were no obvious treatment-related changes in other in-life parameters including mortality, clinical appearance and functional observations. At 1000 ppm, body weight and food consumption were considered unaffected by treatment.

Haematology: Some treatment related but non-adverse effects were noted at 6000 ppm. Minor haematological changes were noted in males and/or females primarily consisting of lower reticulocyte counts (approximately 40% lower than controls), and less pronounced changes consisting of higher red blood cell counts, and haematocrit, mean corpuscular volume and mean corpuscular haemoglobin. These changes were indicative on an effect on red blood cell turn over, and could be related to the morphological changes in the spleen (decreased incidence and severity of extramedullary haematopoiesis, and lower spleen weights in females). Marginally lower haematocrit recorded for males at 3000 ppm had no morphological correlates and at 1000 ppm, haematological parameters were considered unaffected by treatment.

Clinical biochemistry: Some non-adverse effects were seen. Increases in alanine and aspartate aminotransferase activity, total bilirubin and bile acids in males and/or females at 6000 ppm could be related to the histopathological liver effects (see the discussion at that section). Other clinical biochemistry changes that were considered to be treatment-related consisted of decreases in total protein and albumin in males at 6000 ppm and females at 3000 and 6000 ppm, higher potassium and urea and lower creatinine and calcium in males at 6000 ppm, and lower cholesterol in females at 6000 ppm. These changes were generally mild in degree and essentially remained within the range considered normal for rats of tis age and strain. Additionally, since these variations had no apparent treatment-related morphological correlates, these were not considered adverse.

Liver effects: At 3000 ppm (females) and 6000 ppm (both sexes), adverse, treatment-related changes in the liver were recorded. These consisted of a combination of peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis. Increases in alanine and aspartate aminotransferase activity, total bilirubin and bile acids in males and/or females at 6000 ppm could be related to the peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis noted in the liver of these males. An increased incidence of peribiliary inflammatory cell infiltrate was also noted in males at 3000 ppm. Based on its minimal severity and the absence of any degenerative changes such as single cell necrosis, this finding in 3000 ppm treated males was considered to be non-adverse. Additionally, two females at 6000 ppm showed slight hepatocellular hypertrophy.

Spleen effects: Non-adverse effects were seen. At 6000 ppm, a decreased incidence and severity of extramedullary haematopoiesis was recorded in the spleen of males and females at 6000 ppm. This correlated with lower spleen weights in females. A few minor haematological changes were noted at 6000 ppm in males and/or females that could be related to these morphological changes in the spleen, and consisted of higher red blood cell counts, and lower reticulocyte counts, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin. None of these haematological changes were however considered adverse, and therefore the morphological changes in the spleen were also not considered adverse.

Thymus effects: Non-adverse effects were seen. At 3000 and 6000 ppm, minimal to slight lymphoid atrophy of the thymus was recorded for females. This correlated with lower thymus weights of females at 3000 and 6000 ppm and with a reduced thymus size in a single female of this group. This lymphoid atrophy was not accompanied by any other indicator of toxicity and was, at this degree, considered non-adverse.

Thyroid effects: Non-adverse effects were seen. At 6000 ppm, a minor increase in follicular cell hypertrophy (up to slight degree) in the thyroid of males was recorded. This was regarded to be an adaptive change and considered to be non-adverse at the incidences and severities recorded. No concurrent treatment-related change in thyroid weight was recorded.

Other organs: No adverse morphological changes were noted in any of the other organs examined in this study.

In conclusion for repeated dose toxicity, the key adverse changes were recorded in the liver at 3000 (females) and 6000 ppm (males and females) characterized by peribiliary inflammation/necrosis. Though a number of other changes are seen and may be treatment related and in view of the mild character were not considered adverse. These effects are covered with a NOAEL set at the low dose for females and the mid dose for males: 1000 and 3000 ppm, respectively. These values corresponds to a mean substance intake of 128 mg/kg bw for females and 215 mg/kg for males. The overall parental NOAEL is therefore 128 mg/kg bw.

Fertility effects:

No adverse effects were seen in males up to 6000 ppm relating to 452 mg/kg bw. Adverse effects were seen in females at a dose where also repeated dose effects were recorded. At 6000 ppm (nominally 400 mg/kg bw for dams), a lower number of implantation sites was recorded versus control 9.9 versus 11.9, without dose relation. No treatment-related changes were noted in any of the other reproductive parameters investigated in this study (i.e. mating, fertility and conception indices, precoital time, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs). The decreased implantation sites can be associated with lower mean body weights of females of 9% at the end of the post-coitum phase.

In conclusion for fertility: No adverse on male fertility was seen up to 452 mg/kg bw (dietary exposure of 6000 ppm). The female LOAEL is 638 mg/kg bw after dietary exposure of 6000 ppm. In the presence of a repeated dose toxicity of 128 mg/kg bw, the overall NOAEL for fertility is set at 386 mg/kg bw. 

Effects on developmental toxicity

Description of key information

Developmental toxicity (OECD TG 422): NOAEL = 386 mg/kg bw (based on a dietary exposure of 3000 ppm).

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 August 2016 - 19 october 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developme ntal Toxicity Screening Test)

Version / remarks:

july 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: The United States Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

july 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

July 2016

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/D evelopmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

May 2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28- day oral toxicity study in rodents

Version / remarks:

July 2000

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)

Version / remarks:

October 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

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

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males: approximately 10 weeks; Females: approximately 12 weeks.
- Weight at study initiation: males 297-334 g; females 197-236 g
- Fasting period before study: no
- Housing: pretest: Females were housed in groups of 5 females/cage in Macrolon plastic cages; premating: Animals were housed in groups of 5 animals/sex/cage in Macrolon plastic cages; mating: Females were caged together with males on a one-to-one-basis in Macrolon plastic cages; postmating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon plastic cages; lactation: Pups were kept with the dam until termination in Macrolon plastic cages. 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.
general: Sterilized sawdust as bedding material and paper as cage-enrichment/nesting material were supplied. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage without cage-enrichment, bedding material, food and water.
- Diet: Free access to prepared diets. During the acclimatization period, animals had free access to standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany). During motor activity measurements, animals did not have access to food for a maximum of 2 hours. The diet was provided in stainless steel containers, covered by a stainless steel grid to prevent spillage. The same diets remained in the food hopper for a maximum of 1 day. On the day of weighing the remaining food in the food hopper, remaining diet was replaced with new diet retained from the freezer acclimated to room temperature conditions for at least 1 hour prior to opening the diet bag.
- Water: Free access to tap-water. During motor activity measurements, animals did not have access to water for a maximum of 2 hours.
- Acclimation period: At least 5 days prior to start of pretest (females) or treatment (males).

DETAILS OF FOOD AND WATER QUALITY:
Diet, water, bedding and cage-enrichment/nesting material evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

ENVIRONMENTAL CONDITIONS (set to maintain)
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 23 August 2016 To: 19 October 2016

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Standard powder rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Storage temperature of food: Diets were kept in the freezer (≤-15°C, for a maximum of 16 days) until use, if not used on the day of preparation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase according to a validated method.
Group Analysis (type of sample)
1 acc (M)
2 acc + hom (TMB)
3 acc (M)
4 acc + hom (TMB)
Duplicate samples were analyzed
Type of sample T=Top, M=Middle, B=Bottom position of container
Analysis acc=accuracy, hom=homogeneity
The accuracy of diet preparations was considered acceptable if the mean measured concentrations was 80-120% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%.
Stability of diet preparations at 500 and 15,000 ppm was determined over 18 days at room temperature and over 16 days in the freezer (≤-15°C) prior to conduct of the dose range finding study, as part of the analytical method development and validation study. Based on these stability results over 8 days at room temperature, additional stability analyses were conducted over 1, 2 and 4 days at room temperature prior to conduct of the dose range finding study.
In addition, random back-up diet samples were taken and stored at ≤-15ºC for possible future analysis. Any remaining samples at finalization of the study report were discarded. Sampling occurred as soon as possible after preparing the diets. If the analytical determinations were not performed on the day of preparation, the samples were stored at ≤-15ºC.

Details on mating procedure:

Details on mating procedure
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum of 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 (how): females were housed individually
with nesting materials

Duration of treatment / exposure:

Males were exposed for 28 days, i.e. 2 weeks prior to mating, during mating, and up to the day prior to scheduled necropsy.
Females that delivered were exposed for 50 - 57 days, i.e. during 2 weeks prior to mating (with the objective of covering at least two complete estrous cycles), the variable time to conception, the duration of the pregnancy and at least 13 days after delivery up to and including the day before scheduled necropsy. Females which failed to deliver healthy offspring were exposed for 41 or 43 days.
Pups were not dosed directly but were potentially exposed to the test item in utero, via maternal milk or from exposure to maternal urine/feces or spilled diet from the food hopper.

Frequency of treatment:

ad libitum

Duration of test:

28 days (males), 50-57 days (females that deliverd), 41-43 days (females which failed to deliver), pups until day 13-15 of lactation

Dose / conc.:

66 mg/kg bw/day (nominal)

Remarks:

equivalent to 1000 ppm dietary exposure

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

equivalent to 3000 ppm dietary exposure

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

equivalent to 6000 ppm dietary exposure

No. of animals per sex per dose:

10

Control animals:

yes, concurrent no treatment

Details on study design:

Doses for the main study were selected based on the results of a dose range finder study. In this study 5 males and 5 females were administered at dietary dose levels of 0, 5000 and 15,000 ppm for 14 days.
At 15,000 ppm, hunched posture was recorded for 2/5 females, reduced bodyweight gain of males and females (10-15%) and reduced food consumption (both sexes) were noted. The relative liver and kidney weights of males were significantly increased and an extended diestrus for 4/5 females were seen. Histopathology at 15,000 ppm showed possible test item related moderate bilateral tubular degeneration in the testes, with cell debris and reduced luminal sperm in the epididymides in 1/5 males, and mucification of the vagina (up to moderate degree) with cellular debris in 4/5 females.
At 5000 ppm, reduced bodyweight gain and mainly in females reduced food consumption were noted on some days, also increased liver weights of males and females were noted.
Based on these results, dose levels of 1000, 3000 and 6000 ppm were selected for the main study.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: 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 PND 1, 4, 7 and 13.

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no treatment related effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (o/n)
- How many animals: all
- Parameters checked according to Guideline

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes (o/n)
- How many animals: all
- Parameters checked according to Guideline

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes, FOB
- Time schedule for examinations: males during week 4 of treatment, females during the last week of lactation (e.g. PND 6-13)
- Dose groups that were examined: 5/sex/dose
- Battery of functions tested: hearing ability, pupillary reflex, static righting reflex, fore-and hind-limb strength, locomotor activity.

IMMUNOLOGY: No

OTHER: estrous cycle determination and general reproduction data.
Determination plasma T4 and TSH levels at necropsy.
Estrous cyclicity (parental animals): yes (14 days prior to treatment, 14 days of treatment and during mating until evidence of mating, and on the day of necropsy)
Sperm parameters (parental animals): Parameters examined in F1 males:
testis weight, epididymis weight., histopatology to examine staging of spermatogenesis from all males of Groups 1 and 4 and all males of Groups 1 and 4 and all males that failed to sire.

Postmortem examinations (parental animals)
GROSS PATHOLOGY: Yes according to guideline.

HISTOPATHOLOGY: Yes according to guideline

Other examinations
Organ weights from 5 selected animals/sex/group: Adrenal glands, Brain, Cowper’s glands,Epididymides, Glans penis, Heart, Kidneys, Levator ani plus bulbocavernosus muscle complex (LABC), Liver, Ovaries, Prostate, Seminal vesicles including coagulating glands, Spleen, Testes, Thymus, Thyroid (including parathyroid if detectable), Uterus (including cervix).
Organ weights from all remaining animals: Cowper’s glands, Epididymides, Glans penis, Levator ani plus bulbocavernosus muscle complex (LABC), Testes, Thyroid.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: No
- Number of late resorptions: No

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No

Mortality/Viability, clinical signs, body weights, sex, anogenital distance, areola/nipple retention, thyroid hormone analysis (PND 4 pups and PND 13-15 pups)

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 ttest) 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.
• The Kruskal-Wallis nonparametric ANOVA test was applied to motoractivity data to determine intergroup differences.
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.

Indices:

Offspring viability indices
Post-implantation survival index (%) = (Total number of offspring born/ Total number of uterine implantation sites) x 100
Post-implantation survival index was expressed as 100% when the number of offspring exceeded the number of implantation sites recorded.
Live birth index (%) = (Number of live offspring on Day 1 after littering/ Total number of offspring born) x 100
Percentage live males at First Litter Check (%) = (Number of live male pups at First Litter Check/ Total 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/ Total number of live pups at First Litter Check) x 100
Viability index (%) = (Number of live offspring on Day 4 before culling/ Number live offspring on Day 1 after littering) x 100
Lactation index (%) = (Number of live offspring on Day 13 after littering/ Number live offspring on Day 4 (after culling)) x 100
Group mean values were calculated from individual litter values.
Sex ratio (percentage males) = (Number of males in litter/ Total number of offspring in litter) x 100

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were noted during daily detailed clinical observations or during weekly arena observations.
Any clinical signs noted among surviving animals during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent doserelated trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female at 1000 ppm was sacrificed moribund on Day 4 of the lactation period following an accidental injury. Following temporary unconsciousness, this animal displayed signs of ill health and deficiency in maternal care. Histopathologically, the cause of moribundity for this animal was considered to be mucosal necrosis in the jejunum, which was supported at necropsy by dark red focus/foci on the jejunum and blood/blood clots in the abdominal cavity.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No treatment-related changes in body weights or body weight gain were noted in males up to and including 3000 ppm and in females at 1000 ppm.
At 6000 ppm, body weights and body weight gain of males and females were lower than controls throughout treatment, achieving a level of statistical significance on most occasions. At the end of treatment, the difference in absolute mean weight to controls was approximately 7% for males. For females, the difference in absolute mean weight to controls was approximately 9% at the end of post-coitum and approximately 16% at the end of lactation. For a few of these females, slight weight loss (ranging from 3 to 8%) was recorded during lactation, and during the first week of the treatment period.
At 3000 ppm, body weights and body weight gain of females were lower than controls during the lactation period only, achieving a level of statistical significance on most occasions. Slight weight loss (2 or 3%) was recorded for a few of these females during lactation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 6000 ppm, a lower mean food consumption before and after allowance for body weight was recorded for females during the post-coitum and most predominantly during the lactation period. The overall mean food intake during lactation was approximately 37% lower than controls.
At 3000 ppm, mean food consumption of females was lower in the second week of the lactation period, but differences from control values were not statistically significant.
No treatment-related changes in food consumption before or after allowance for body weight were noted in males up to 6000 ppm and in females at 1000 ppm.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in haematology parameters distinguished treated from control animals:
• Elongated prothrombin time (PT) in males and females at 6000 ppm.
• Higher red blood cell counts in females at 6000 ppm.
• Lower reticulocyte counts in males and females at 6000 ppm (not statistically significant for males).
• Lower haematocrit in males at 3000 and 6000 ppm.
• Lower mean corpuscular volume (MCV) in females at 6000 ppm.
• Lower mean corpuscular haemoglobin (MCH) in females at 6000 ppm.
No treatment-related changes in haematology parameters were noted in males and females treated at 1000 ppm. Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend and/or remained within the range considered normal for rats of this age and strain.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals from control animals:
• Higher alanine aminotransferase activity (ALAT) in males at 6000 ppm.
• Higher aspartate aminotransferase activity (ASAT) in males at 6000 ppm.
• Lower total protein in males at 6000 ppm, and in females at 3000 and 6000 ppm.
• Lower albumin in males at 6000 ppm, and in females at 3000 and 6000 ppm.
• Higher total bilirubin in females at 3000 and 6000 ppm.
• Higher urea in males at 6000 ppm.
• Lower creatinine in males at 6000 ppm.
• Lower cholesterol in females at 6000 ppm.
• Higher bile acids in males at 6000 ppm and in females at 3000 and 6000 ppm (not statistically signif
icant for females; higher mean at 6000 ppm was mainly due to one female.
• Higher potassium in males at 6000 ppm.
• Lower calcium in males at 6000 ppm.
No treatment-related changes in clinical biochemistry parameters were noted in males treated up to and including 3000 ppm and in females treated at 1000 ppm. The apparent higher mean alkaline phosphatase activity (ALP) in females at 3000 and 6000 ppm was due to a high interindividual variation which could not be attributed to treatment-related histopathological changes. As such these were considered to have occurred by change and to be of no toxicological significance. Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and treated animals.
Motor activity data showed a similar habituation profile with a decreasing trend in activity over the duration of the test period for all groups.
For males treated at 6000 ppm an apparent trend towards lower mean values for total movements and ambulations was recorded. This was not considered toxicologically relevant since these variations were not statistically significant and remained within the range considered normal for rats of this age and strain, and habituation profile of these animals was similar to that encountered for control animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant test item-related higher liver and kidney weights (relative to body weight) were noted in the 6000 ppm treated group males and test item-related significant lower thymus and spleen weights (absolute) were noted in the 6000 ppm treated females. For females, the thymus weights showed a dose-related decrease and for males there was an apparent decrease in thymus weights at 6000 ppm (see table in 'other information"). The decreased thymus weights in females treated at 3000 and 6000 ppm correlated microscopically with lymphoid atrophy. The decreased spleen weights in females treated at 6000 ppm correlated microscopically with decreased hematopoiesis. There was no microscopic correlate for the other organ weights changes. Some other organ weight differences were statistically significant when compared to the control group but were considered to be the result of a test item-related decrease in body weight.
There were no other test item-related organ weight changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item-related macroscopic findings were present in the thymus of a single female treated at 6000 ppm in the form of reduced size. This correlated microscopically with lymphoid atrophy.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test item-related microscopic findings were present in the liver of males and females treated at 3000 and 6000 ppm, in the spleen of males and females treated at 6000 ppm, in the thymus of females treated at 3000 and 6000 ppm, and in the thyroid gland of males treated at 6000 ppm, and are summarized in text tables A, B and C (see "other information").
The following changes were observed in the liver:
• Inflammatory cell infiltrate peribiliary was present at increased incidence and severity in males and females treated at 3000 and 6000 ppm up to moderate degree.
• Single cell necrosis peribiliary was present in females treated at 3000 and 6000 ppm up to moderate degree and in males treated at 6000 ppm at minimal degree.
• Hepatocellular hypertrophy was present in females treated at 6000 ppm at slight degree.
In the spleen, a decreased incidence and severity of extramedullary hematopoiesis was present in males and females treated at 6000 ppm. For females this correlated with the decreased spleen weights at 6000 ppm.
In the thymus, lymphoid atrophy was present in females treated at 3000 and 6000 ppm up to slight degree. For a single female at 6000 ppm this correlated with the macroscopic finding reduced in size. This correlated with the lower thymus weights in females at 6000 ppm.
In the thyroid gland, follicular cell hypertrophy was present at increased incidence and severity in males treated at 6000 ppm up to slight degree.
The remainder of the recorded microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test item-related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations.

Histopathological findings: neoplastic:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

At 6000 ppm, a lower post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was recorded at 70% at 6000 ppm versus 95% in the control group. One female at 6000 ppm had 10 implantations but no offspring and another female of this group had 7 implantations and delivered only one live pup, resulting in survival percentages of 0% and 14%, respectively. To a lesser extent, post-implantation survival was also lower in 2 other females (approximately 70% survival). Post-implantation survival in the remaining five females at 6000 ppm was normal.
Also, females at 6000 ppm had a lower mean number of living pups at first litter check (7.6, versus 11.2 in the control group). The post-implantation survival indices at 1000 and 3000 ppm (88% and 87%, respectively) were considered not to be affected by treatment. One pregnant female at 1000 ppm and one pregnant female at 6000 ppm had implantation sites only. All other females delivered live offspring.
For one female at 1000 ppm (sacrificed moribund on lactation Day 4), the number of pups was slightly higher than the number of implantation sites. This was considered to be due to normal resorption of these areas as this enumeration was performed on Day 4 of lactation.

Early or late resorptions:

not specified

Dead fetuses:

no effects observed

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices in the different groups ranged from 98 to 100%. One control female and one female dosed 6000 ppm had a dead pup at first litter check. This incidental pup mortality was unrelated to treatment with the test item.

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.DescriptionIncidenceAndSeverityEffectsOnPregnancyDuration): Gestation index and duration of gestation were not affected by treatment. A total of two females each at 1000 and 6000 ppm had no offspring, resulting in a gestation index of 89% at both these dose levels. One female each at 1000 and 6000 ppm had implantation sites only, and one female each at 1000 and 6000 ppm were not pregnant. The incidence of absence of offspring at these dose levels showed no relationship to the dose and was considered to be within the range considered normal for rats of this age and strain. As such, this was not considered to be related to treatment.

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

A total of two females each at 1000 and 6000 ppm had no offspring, resulting in a gestation index of 89% at both these dose levels. One female each at 1000 and 6000 ppm had implantation sites only, and one female each at 1000 and 6000 ppm were not pregnant. The incidence of absence of offspring at these dose levels showed no relationship to the dose and was considered to be within the range considered normal for rats of this age and strain. As such, this was not considered to be related to treatment.

Other effects:

no effects observed

Description (incidence and severity):

No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed among the surviving animals.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other: dietary concentration was 1000 ppm

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 3000 and 6000 ppm, lower mean body weights were recorded for male and female pups from PND 1 onwards. At PND 1, mean body weights were decreased by about 5% (3000 ppm) or 10% (6000 ppm), and at PND 13 these differences had increased to 13% (3000 ppm) and 39% (6000 ppm). The differences were statistically significant on most occasions in the 6000 ppm group and at PND 13 in the 3000 ppm group.
Pup body weights at 1000 ppm were not considered affected by treatment.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): not specified

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment. The live birth indices in the different groups ranged from 98 to 100%.
One control female and one female dosed 6000 ppm had a dead pup at first litter check. This incidental pup mortality was unrelated to treatment with the test item.

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

not specified

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

The viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1 was not affected by treatment. The viability indices in the different groups ranged from 90% (1000 ppm) to 98% (3000 and 6000 ppm) or 100% (control group). Post-natal loss up to PND 4 was limited to one pup at 1000 ppm which went missing on PND 2, two pups at 3000 ppm that went missing on PND 3 or 4, and one pup at 6000 ppm which went missing on PND 2. The missing pups were most likely cannibalised. No toxicological relevance was attributed to these missing pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.
The statistically significantly higher total number of pups lost between PND 1-4 at 1000 ppm can be explained by the premature sacrifice of one female together with her eight healthy pups. The moribund condition of this female was not related to treatment (see section 7.2.1 Mortality).

External malformations:

effects observed, treatment-related

Description (incidence and severity):

At 6000 ppm, all pups of two litters had a lean appearance. A lean appearance was also noted up to the day prior to scheduled sacrifice in the single pup of one female dosed 6000 ppm and most likely this pup was lean at sacrifice (inadvertently, macroscopic findings of this pup were not recorded). Additionally, all pups of one litter had a gastrointestinal tract distended with gas. These findings correlated with the lower pup
body weights at 6000 ppm and were considered to be related to the treatment.
No other macroscopic findings were noted among pups that were considered to be related to treatment. The incidental macroscopic findings noted in the two pups found dead at first litter check (one pup at 0 and one pup at 6000 ppm) were unrelated to treatment as they represented normal background findings and occurred incidentally. No macroscopic findings were noted in the pups of one female dosed 1000 ppm which was sacrificed moribund during lactation.

Skeletal malformations:

not examined

Visceral malformations:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related clinical signs were noted in all pups of three females treated at 6000 ppm ( 8, 8 and 1 surviving pups, respectively). A (very) lean appearance was noted for the surviving pups of 2 females at PND 13 and/or 14, and in the pup of one female from PND 4 onwards. The lean appearance correlated with the lower pup body weights at 6000 ppm. Additionally, the pups of two of these females were dehydrated at PND 13 and 14, and the pup of one of these females had little milk in the stomach at PND 7, 8 and 10.
No other clinical signs occurred among pups that were considered to be related to treatment. The nature and incidence of the clinical signs observed incidentally at 1000 and 3000 ppm remained within the range considered normal for pups of this age, and were therefore considered to be of no toxicological relevance.

Anogenital distance (absolute and normalized for body weight) in male and female pups was not affected by treatment.
Treatment up to and including 6000 ppm had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Serum T4 levels in male and female PND 13-15 pups were not affected by treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

fetal/pup body weight changes

changes in litter size and weights

other: lower post-implantation survival

Remarks on result:

other: dietary concentration was 3000 ppm

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

The concentrations analyzed in the diets of Groups 2 - 4 (1000, 3000 and 6000 ppm, respectively) were in agreement with target concentrations with mean accuracies between 106 and 109).

No test item was detected in the Group 1 diet. The diets of Group 2 (1000 ppm) and Group 4 (6000 ppm) were homogeneous, with coefficient of variation ranging from 2.4 to 2.8.

Analysis of the diets at 500 ppm (low) and 15000 ppm (high) after storage yielded a relative differences of ≤ 10% (actual data -3.1% to -8.5%) when stored for 1 or 2 days and > 10% (actual data -19% and -16% in low and high dose groups, espectively) when stored for 4 days. Based on this, the diets were considered to be stable during storage at room temperature under normal laboratory light conditions

for 2 days.

The mean daily intake of the test item per kg body weight during the different phases of the study is given in the table below (mg/test item/kg bw/day, overall group means in the indicated period)).

Mean of means of all periods, weighed for number of measurement intervals per period:

Males: ((14 x mean premating) + (12 x mean mating)) / 26

Females: ((14 x mean premating) + (21 x mean post-coitum) + (13 x mean lactation)) / 48

	Group 2 1000 ppm	Group 3 3000 ppm	group 4 6000 ppm
Males
Pre-mating	75	226	466
Mating	67	203	435
Mean of means	71	215	452
Females
Pre-mating	73	226	416
Post-coitum	109	324	592
Lactation	218	659	950
Mean of means	128	386	638

Organ weights

Mean Percent Liver, Thymus, Spleen and Kidneys Weight Differences from Control Group ( *: P<0.05, **: P<0.01)

	Males			Females
Dose level (ppm)	1000	3000	6000	1000	3000	6000
Liver absolute	0	9	2	-1	13	-16
Liver relative to BW	1	8	13**	1	21	1
Thymus absolute	13	-1	-26	-1	-16	-30*
Thymus relative to BW	12	-2	-18	-4	-9	-17
Spleen absolute	18	11	-3	-3	-10	-22*
Spleen relative to BW	17	10	7	-1	-2	-6
Kidneys absolute	-2	11	4	-3	-1	-8
Kidneys relative to BW	-3	9	15*	-3	7	11

Microscopic examination

Table ASummary Test Item-Related Microscopic Findings – Spleen and Liver – Both Sexes

(a )= Number of tissues examined from each group.

	Males				Females
Dose level (ppm)	0	1000	3000	6000	0	1000	3000	6000
Liver	5	5	5	5	5	5	5	5
Inflammatory cell infiltrate peribiliary
Minimal	1	2	4	-	-	-	3	-
Slight	-	-	-	3	-	-	2	4
Moderate	-	-	-	2	-	-	-	1
Single cell necrosis peribiliary
Minimal	-	-	-	2	-	-	3	2
Slight	-	-	-	-	-	-	-	3
Moderate	-	-	-	-	-	-	1	-
Hepatocellular hypertrophy
Slight	-	-	-	-	-	-	-	2
Spleen	5	5	5	5	5	5	5	5
Hematopoiesis, extramedullary
Minimal	3	4	4	-	4	4	2	1
Slight	1	-	1	-	1	-	2	-

Table B. Summary Test Item-Related Microscopic Findings – Thymus - Females Only

(a) = Number of tissues examined from each group.

	Females
Dose level (ppm)	0	1000	3000	6000
Thymus(a)	5	5	5	5
Atrophy lymphoid
Minimal	-	-	2	3
Slight	-	-	-	1

Table CSummary Test Item-Related Microscopic Findings – Thyroid Gland - Males Only

(a) = Number of tissues examined from each group

	Males
Dose level (ppm)	0	1000	3000	6000
Thyroid gland(a)	5	5	5	5
Hypertrophy follicular cell
Minimal	2	3	3	2
Slight	-	-	-	2

Conclusions:

In conclusion, treatment with the substance dosed via diet at dose levels 1000, 3000 and 6000 ppm resulted in a NOAEL for parental toxicity of 1000 ppm (corresponding to a mean test item intake of 128 mg/kg bw/day in females) based on histopathological changes in the liver characterized by the combination of peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis at 3000 ppm in females and 6000 ppm in males and females. The NOAEL for developmental toxicity in this study is 3000 ppm (corresponding to a mean daily test item intake of 215 mg/kg bw/day in males and 386 mg/kg bw/ day in females), based on lower body weights and body weight gain of pups at 6000 ppm, with lower litter size and post-implantation survival.

Executive summary:

In the executive summary of the developmental toxicity also the repeated dose toxicity information is inclued to present the context of the information. Fertilty is presented in the fertility section.

Bicyclononalactone repeated dose and developmental toxicity

For Bicyclononalactone a combined repeated dose toxicity study with the reproductive/developmental screening test according to OECD 422 was performed with rat. Based on a range finding test the following concentrations in the diet were used, with dose groups 0, 1000, 3000 and 6000 ppm, aiming at doses of 66, 200 and 400 mg/kg bw. The following repeated dose toxicity parameters were recorded; mortality, clinical signs, functional observations and locomotor activity, body weight and food consumption, haematology parameters and organ weights, and macroscopy and histopathology on a selection of tissues. In addition thyroid hormone T4 (F0 males at the end of treatment and PND 13 -15 pups) were measured. The parameters assessed for fertility and developmental toxicity are described at the respective sections. The doses were confirmed by analysis.

Analysis of the test substance intakeshowed that the dietary concentrations of 1000, 3000 and 6000 ppms resulted in doses for males: 71, 215 and 452 mg/kg bw and for females 128, 386, 638 mg/kg bw, respectively. 

Clinical signs and FOB: There were no treatment-related changes in clinical appearance and functional observations.

Body weight and food consumption: Lower body weight and food consumption was recorded at 3000 and 6000 ppm. At 6000 ppm, body weight and body weight gain was lower in males and females throughout the treatment period with occasional slight weight loss for females during the first week of treatment and during lactation. At the end of treatment, mean body weight was approximately 7 and 16% lower than controls for males and females, respectively. For females, this was accompanied by a lower food consumption during the post-coitum period and, more markedly (nearly 40% difference from controls), during the lactation period. Since lower body weight gain was also noted in males, a direct test item related effect was considered likely. At 3000 ppm, body weight and body weight gain was lower for females during the lactation period, along with lower food intake in the second week of the lactation period. Despite the lower body weights and food intake at 3000 and 6000 ppm, animals appeared in a good health condition based on absence of treatment-related changes in other in-life parameters including mortality, clinical appearance and functional observations. There were no obvious treatment-related changes in other in-life parameters including mortality, clinical appearance and functional observations. At 1000 ppm, body weight and food consumption were considered unaffected by treatment.

Haematology: Some treatment related but non-adverse effects were noted at 6000 ppm. Minor haematological changes were noted in males and/or females primarily consisting of lower reticulocyte counts (approximately 40% lower than controls), and less pronounced changes consisting of higher red blood cell counts, and haematocrit, mean corpuscular volume and mean corpuscular haemoglobin. These changes were indicative on an effect on red blood cell turn over, and could be related to the morphological changes in the spleen (decreased incidence and severity of extramedullary haematopoiesis, and lower spleen weights in females). Marginally lower haematocrit recorded for males at 3000 ppm had no morphological correlates and at 1000 ppm, haematological parameters were considered unaffected by treatment.

Clinical biochemistry: Some non-adverse effects were seen. Increases in alanine and aspartate aminotransferase activity, total bilirubin and bile acids in males and/or females at 6000 ppm could be related to the histopathological liver effects (see the discussion at that section). Other clinical biochemistry changes that were considered to be treatment-related consisted of decreases in total protein and albumin in males at 6000 ppm and females at 3000 and 6000 ppm, higher potassium and urea and lower creatinine and calcium in males at 6000 ppm, and lower cholesterol in females at 6000 ppm. These changes were generally mild in degree and essentially remained within the range considered normal for rats of tis age and strain. Additionally, since these variations had no apparent treatment-related morphological correlates, these were not considered adverse.

Liver effects: At 3000 ppm (females) and 6000 ppm (both sexes), adverse, treatment-related changes in the liver were recorded. These consisted of a combination of peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis. Increases in alanine and aspartate aminotransferase activity, total bilirubin and bile acids in males and/or females at 6000 ppm could be related to the peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis noted in the liver of these males. An increased incidence of peribiliary inflammatory cell infiltrate was also noted in males at 3000 ppm. Based on its minimal severity and the absence of any degenerative changes such as single cell necrosis, this finding in 3000 ppm treated males was considered to be non-adverse. Additionally, two females at 6000 ppm showed slight hepatocellular hypertrophy.

Spleen effects: Non-adverse effects were seen. At 6000 ppm, a decreased incidence and severity of extramedullary haematopoiesis was recorded in the spleen of males and females at 6000 ppm. This correlated with lower spleen weights in females. A few minor haematological changes were noted at 6000 ppm in males and/or females that could be related to these morphological changes in the spleen, and consisted of higher red blood cell counts, and lower reticulocyte counts, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin. None of these haematological changes were however considered adverse, and therefore the morphological changes in the spleen were also not considered adverse.

Thymus effects: Non-adverse effects were seen. At 3000 and 6000 ppm, minimal to slight lymphoid atrophy of the thymus was recorded for females. This correlated with lower thymus weights of females at 3000 and 6000 ppm and with a reduced thymus size in a single female of this group. This lymphoid atrophy was not accompanied by any other indicator of toxicity and was, at this degree, considered non-adverse.

Thyroid effects: Non-adverse effects were seen. At 6000 ppm, a minor increase in follicular cell hypertrophy (up to slight degree) in the thyroid of males was recorded. This was regarded to be an adaptive change and considered to be non-adverse at the incidences and severities recorded. No concurrent treatment-related change in thyroid weight was recorded.

Other organs: No adverse morphological changes were noted in any of the other organs examined in this study.

In conclusion for repeated dose toxicity, the key adverse changes were recorded in the liver at 3000 (females) and 6000 ppm (males and females) characterized by peribiliary inflammation/necrosis. Though a number of other changes are seen and may be treatment related and in view of the mild character were not considered adverse. These effects are covered with a NOAEL set at the low dose for females and the mid dose for males: 1000 and 3000 ppm, respectively. These values corresponds to a mean substance intake of 128 mg/kg bw for females and 215 mg/kg for males. The overall parental NOAEL is therefore 128 mg/kg bw.

Developmental results:

Adverse effects were seen at doses where also repeated dose effects were recorded. At 6000 ppm, a lower total number of living pups at first litter check and litter size (mean number of living pups at first litter check; 7.6, versus 11.2 in the control group) were recorded. The lower litter size could partly be explained by the lower number of implantation sites (9.9, versus 11.9 in the control group) and partly by a lower post-implantation survival at 6000 ppm (70%, versus 95% in the control group). This lower value at 6000 ppm was due to lower post-implantation survival in four out of the nine pregnant females in this group. The number of dead pups at first litter check and the live birth index at 6000 ppm were not affected by treatment.

At 3000 and 6000 ppm, lower mean body weights were recorded for male and female pups from PND 1 onwards, which became increasingly pronounced as treatment progressed; at PND 13 the weight difference to control means was approximately 13% at 3000 ppm and 39% at 6000 ppm. Clinical and macroscopic examination of pups at 6000 ppm showed a (very) lean appearance for pups of three females treated at 6000 ppm, which correlated with markedly lower pup body weights at 6000 ppm. Additional clinical and macroscopic findings noted in these pups consisted of dehydration, little milk in the stomach and gastrointestinal tract distended with gas. No clear relationship between body weight gain and food intake of dams during lactation and body weight development of their respective litters was apparent. However, it cannot be excluded that at least the lower pup body weights may in part be related to the lower body weight and food intake of the dams, which was 13% and ca. 40%, respectively lower compared to the controls at the high dose. Overall, the effects on litter size, post-implantation survival and pup body weight with associated clinical signs were considered to represent a treatment-related effect on pup development and were therefore considered adverse at 6000 ppm. 

Other developmental effects: No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and the early postnatal pup developmental parameters mortality, anogenital distance (PND 1), areola/nipple retention (PND 13 males), and serum concentration of the thyroid hormone T4 (PND 13-15).

In conclusion for developmental toxicity: In the presence of a repeated dose NOAEL of 128 mg/kg bw, the LOAEL based on the developmental toxicity effects seen is 6000 ppm (638 mg/kg bw) resulting in a NOAEL of 3000 ppm, being 386 mg/kg bw.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

386 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The database contains information according to OECD TG 422 under GLP. This information is reliable and adequate for covering this endpoint.

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 section at fertility both repeated dose and fertility is presented. In this section for the repeated dose toxicity is presented above and not repeated here.

Developmental results:

Adverse effects were seen at doses where also repeated dose effects were recorded. At 6000 ppm, a lower total number of living pups at first litter check and litter size (mean number of living pups at first litter check; 7.6, versus 11.2 in the control group) were recorded. The lower litter size could partly be explained by the lower number of implantation sites (9.9, versus 11.9 in the control group) and partly by a lower post-implantation survival at 6000 ppm (70%, versus 95% in the control group). This lower value at 6000 ppm was due to lower post-implantation survival in four out of the nine pregnant females in this group. The number of dead pups at first litter check and the live birth index at 6000 ppm were not affected by treatment.

At 3000 and 6000 ppm, lower mean body weights were recorded for male and female pups from PND 1 onwards, which became increasingly pronounced as treatment progressed; at PND 13 the weight difference to control means was approximately 13% at 3000 ppm and 39% at 6000 ppm. Clinical and macroscopic examination of pups at 6000 ppm showed a (very) lean appearance for pups of three females treated at 6000 ppm, which correlated with markedly lower pup body weights at 6000 ppm. Additional clinical and macroscopic findings noted in these pups consisted of dehydration, little milk in the stomach and gastrointestinal tract distended with gas. No clear relationship between body weight gain and food intake of dams during lactation and body weight development of their respective litters was apparent. However, it cannot be excluded that at least the lower pup body weights may in part be related to the lower body weight and food intake of the dams, which was 13% and ca. 40%, respectively lower compared to the controls at the high dose. Overall, the effects on litter size, post-implantation survival and pup body weight with associated clinical signs were considered to represent a treatment-related effect on pup development and were therefore considered adverse at 6000 ppm. 

Other developmental effects: No treatment-related changes were noted in any of the other developmental parameters investigated in this study (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care and the early postnatal pup developmental parameters mortality, anogenital distance (PND 1), areola/nipple retention (PND 13 males), and serum concentration of the thyroid hormone T4 (PND 13-15).

In conclusion for developmental toxicity: In the presence of a repeated dose NOAEL of 128 mg/kg bw, the LOAEL based on the developmental toxicity effects seen is 6000 ppm (638 mg/kg bw) resulting in a NOAEL of 3000 ppm, being 386 mg/kg bw.

Justification for classification or non-classification

The parental NOAELin this study is set at 128 mg/kg bw (corresponding to a dietary exposure of 1000 ppm) based on histopathological changes in the liver characterized by the combination of peribiliary inflammatory cell infiltrate and peribiliary single cell necrosis at 383 mg/kg bw in females (3000 ppm ) and 452 and 683 mg/kg bw for males and females, respectively.

The NOAEL for fertility is set at 383 mg/kg bw (corresponding to a dietary exposure of 3000 ppm in females, based on a reduced number of implantation sites at 683 mg/kg bw (dietary exposure 6000 ppm). No adverse male fertility effects were seen.

The NOAEL for developmental toxicity is set at 383 mg/kg bw (corresponding to a dietary exposure of 3000 ppm of females), based on lower body weights and body weight gain of pups at 683 mg/kg bw (dietary exposure 6000 ppm). The observed reproduction toxicity and developmental toxicity were in the presence of maternal toxicity. Based on the observed effects, classification of the substance is not warranted according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No.1272/2008 and its amendments.