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

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

reproduction and developmental screening

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

july 2016

Deviations:

no

Test guideline 2

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

Test guideline 3

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2016

Deviations:

no

Test guideline 4

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

July 2000

Deviations:

no

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 6

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

October 2008

Deviations:

no

Test guideline 7

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

GLP compliance:

yes

Limit test:

no

Test material

Test material information

1

Reference substance name:

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

EC Number:

224-623-4

EC Name:

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

Cas Number:

4430-31-3

Molecular formula:

C9H14O2

IUPAC Name:

octahydro-2H-1-benzopyran-2-one

Test material form:

liquid

Test animals

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 toxicity 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; pre-mating: 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

Administration / exposure

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as it is a possible route of human exposure during manufacture, handling or use of the test item.The amount of test item incorporated into the diet was kept at a constant level in terms of ppm, throughout the study period.
The actual test item intake was estimated based on the body weight and food consumption values.

Vehicle:

unchanged (no vehicle)

Details on oral 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.

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

66 mg/kg bw/day (nominal)

Remarks:

Converted from dietary exposure of 1000 ppm

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Converted from dietary exposure of 3000 ppm

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw/day (nominal)

Remarks:

Converted from the dietary exposure of 6000 ppm

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.

Examinations

Observations and examinations performed and frequency:

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 of plasma T4 and FSH levels at necropsy.

Sacrifice and pathology:

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

Examinations of pups (see section 7.8.2)

Statistics:

The following statistical methods were used to analyse the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• 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.

Results and discussion

Results of examinations

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 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

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

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.

Effect levels

Key result

Dose descriptor:

NOAEL

Remarks:

parental

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Remarks on result:

other:

Remarks:

Females and males had different test substance intake. At 1000 ppm the females showed intake of 128 mg/kg bw, while males at 1000 ppm showed 71mg/kg bw. The NOAEL for females was 1000 ppm (128 mg/kg bw, while for males it was 3000 ppm (215 mg/kg bw). Therefore it was considered justified to have the overall NOAEL based on females.

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

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 (a)	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 (a)	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

Applicant's summary and conclusion

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.

Executive summary:

The executive summary of the repeated dose toxicity is presented here. The fertiltiy and developmental toxicity are presented at the reproductive toxicity records.

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.