Alkanes, C10-13-iso-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

May 15, 2017 - December 27, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 29, 2016

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Alkanes, C10-13-iso-

EC Number:

271-366-9

EC Name:

Alkanes, C10-13-iso-

Cas Number:

68551-17-7

Molecular formula:

C10H22 - C13H28

IUPAC Name:

Alkanes, C10-13-iso

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Japan, Inc.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks
- Weight at study initiation: 293 to 336 g in males and 196 to 241 g in females
- Housing: Metal bracket cages with wire mesh floors; Two animals per cage during quarantine and acclimatization period, 1 after group assignment, 1 male and 1 female during mating period, 1 female during gestation period, and 1 litter during lactation period
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 20 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23
- Humidity (%): 43 to 55
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): artificial lighting for 12 hrs (from 8:00 to 20:00)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test article was accurately weighed for each dose level, and the vehicle (control article) was added to achieve the prescribed concentration, and the solution was stirred with a stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test article is insoluble in aqueous solution
- Lot/batch no. (if required): V6N9339

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: 1 female during gestation period, and 1 litter during lactation period

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

the content in the dosing solution which was analyzed by GC-FID corresponded with the nominal concentrations, the RSD (relative standard deviation) were below 15%.

Duration of treatment / exposure:

Males:
Main study group: for 28 days from 14 days before the start of mating
Satellite group: for 28 days

Females:
Main study group: for 14 days before the start of mating and during mating period until successful mating
Females with successful mating: during gestation period until lactation day 13
Satellite group: for 51 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

12 male / 12 female for main group; 5 male / 5 female for Satellite group

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Males: before the start of administration and on administration days 7, 14, 21, 28 (Main study group and Satellite group), and recovery days 7 and 14 (Satellite group)
Females: before the start of administration and on administration days 7, 14, 21, 28, 35, 42, and 49 (Main study group and Satellite group), and recovery days 7 and 14 (Satellite group)

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: before administration on administration days 1, 4, 7, 14, 21, and 28 (Main study group and Satellite group) and on recovery days 1, 7, and 14 (Satellite group), and the day of necropsy (Main study group and Satellite group).
Females in the Main study group: before administration on administration days 1, 4, 7, and 14, and before administration on gestation days 0, 7, 14, and 20, and before administration on lactation days 0, 4, 7 and 13, and day 14 after delivery which was the day of necropsy.
Females in the Satellite group: before administration on administration days 1, 4, 7, 14, 21, 28, 35, 42, and 49 and on recovery days 1, 7, and 14, and the day of necropsy.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER: Functional observations; Hematology; Blood biochemistry; Urinalysis.

Oestrous cyclicity (parental animals):

Yes

Sperm parameters (parental animals):

Parameters examined in male parental generations:
testis weight, epididymis weight and seminal vesicles weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

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, other: serum T4 concentration, necropsy.

GROSS EXAMINATION OF DEAD PUPS: Yes

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after administration day 28
- Maternal animals: All surviving animals on day 14 parturition

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

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were prepared for microscopic examination and weighed:
Brain, heart, liver, kidneys, testes, epididymides, and seminal vesicles (with coagulating glands);
Pituitary glands, thyroids (with parathyroids), spleen, thymus, adrenals, prostate, ovaries, and uterus

Postmortem examinations (offspring):

SACRIFICE
- All offspring were sacrificed at 14 days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

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

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid of 1 male and 1 female per litter were prepared for microscopic examination and weighed, respectively.

Statistics:

Statistical analyses were performed using a computer system (MiTOX).
Grip strength, motor activity, body weight, body weight gain, food consumption, urine volume, hematology, blood biochemistry (except T4), absolute and relative organ weight, length of estrous cycle, the numbers of estrus, number of days required for copulation, number of implantations, offspring delivered, live newborns and dead newborns, birth index, gestation period, body weight of offspring on postnatal days 0 and 4, viability index on postnatal day 4, and incidence of offspring with external anomalies were analyzed by the Bartlett test for homogeneity of variances. For serum T4 concentration, viability index on postnatal days 0 and 13, body weight of offspring on postnatal days 7 and 13, number of papillae, and anogenital distance were separately analyzed by the same statistical procedures as described above using a statistical analysis system (Sanken System Co., Ltd.) or SAS system (SAS Institute Inc.).
Detailed clinical observations, reactivity to environmental stimuli, and results of urinalysis except urine volume were analyzed by the Kruskal-Wallis test.
The incidence of abnormal estrous cycles, copulation index, fertility index, gestation index, sex rate of live offspring on postnatal day 0, and incidence of dams with offspring showing external anomalies, and the results of histopathology (in the Main study groups and the Satellite groups) were analyzed by Fisher's exact probability test.

Reproductive indices:

Copulation index (%) = number of animals with successful copulation/number of animals used for mating × 100
Fertility index (%) = number of animals that impregnated a female or were pregnant/number of animals with successful copulation × 100
Gestation index (%) = number of females with normal parturition/number of pregnant females × 100

Offspring viability indices:

Birth index (%) = number of live offspring on postnatal day 0/number of implantations × 100
Sex rate (%) of live offspring = number of live male offspring/ (number of live male offspring + number of live female offspring) × 100
Incidence of offspring with external anomalies (%) = (number of live offspring with external anomalies / number of live offspring examined) × 100
Incidence of dams with offspring showing external anomalies = number of dams having offspring with external anomalies / number of dams that delivered
Viability index (%) on postnatal day 0 (or 4, 13) = number of live offspring on postnatal day 0 (or 4, 13) /number of implantations × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg bw/day group, soil of perigenital fur was noted in 1 male on administration day 4 and 5 and in 1 female on administration day 6. No abnormalities were noted for all other groups.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Administration period
Males: Main study group and Satellite group
No significant differences were noted in body weight or body weight gain in the 10 or 100 mg/kg group compared with the control group. In the 1000 mg/kg group, body weight on administration days 21 and 28, and body weight gain during administration period were significantly lower than those in the control group.
Females: Main study group
No significant differences were noted in body weight or body weight gain in the 10 or 100 mg/kg group compared with the control group. In the 1000 mg/kg group, no significant differences were noted in body weight or body weight gain during pre-mating period. During breeding period, although no significant differences were noted in body weight gain, body weight on gestation day 7 and 14, and throughout the lactation period was significantly lower than that in the control group.
Females: Satellite group
In the 1000 mg/kg group, no significant differences were noted in body weight or body weight gain compared with the control group.

Recovery period
Males: Satellite group
In the 1000 mg/kg group, no significant differences were noted in body weight or body weight gain compared with the control group.
Females: Satellite group
In a part of the females (animal Nos. 50653 and 50655) in the 1000 mg/kg group, body weight was decreased during the recovery period, and the means tended to be low with no significant differences.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Main study group and satellite group of males:
In the 100 mg/kg group, food consumption on administration days 4, 7, and 14 was significantly lower than that in the control. This change was toxicologically insignificant because no changes were noted in body weight. In the 1000 mg/kg group, food consumption on administration days 4 and 7 was significantly lower than that in the control group. In this group, a significantly high value was noted on administration day 28.
Main study group of females:
No significant differences were noted in the 10 or 100 mg/kg group compared with the control group. In the 1000 mg/kg group, food consumption on administration days 4 and 7 tended to be low or significantly low. During gestation or lactation period, no significant differences were noted.
Satellite group of females:
In the 1000 mg/kg group, food consumption on administration days 4 and 7 tended to be low or significantly low compared with the control group. In this group, significantly high values were noted on administration days 35 and 49.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of administration period
Males: Main study group
No significant differences were noted in any parameter in the 10 or 100 mg/kg group compared with the control group. In the 1000 mg/kg group, APTT was significantly prolonged, and PT tended to be prolonged in 2 males (animal Nos.
10404 and 10405). In this group, RBC and MCHC were significantly low and platelet count was significantly high, both of which were considered not toxicologically significant because these were slight changes and no changes were
noted in the reticulocyte count, histopathological examination revealed no changes indicative of increased hematopoiesis or bleeding tendency, and no changes occurred in HGB or MCV.
Females: Main study group (day 14 after delivery)
In the 10 mg/kg group, MCV was significantly lower than that in the control group, which was considered unrelated to the test article administration because this change was not dose-related. No changes were noted in any parameter in the 100 mg/kg group. In the 1000 mg/kg group, PT was significantly shortened, which was not considered toxicologically significant because no changes occurred in APTT and shortening is a change of low clinical significance.

At the end of recovery period
Males: Satellite group (recovery day 15)
In the 1000 mg/kg group, reticulocyte count was significantly higher than that in the control group, which was considered unrelated to the test article administration because this change was not observed at the end of the administration period, and no changes occurred in RBC or HGB.
Females: Satellite group (recovery day 15)
In the 1000 mg/kg group, platelet count was significantly higher than that in the control group, which was considered unrelated to the test article administration because histopathological examination revealed no changes indicative of increased hematopoiesis, and no changes related to hematopoiesis and inflammation occurred in any parameter of the hematopoietic system such as reticulocyte count and WBC, and was not observed at the end of the administration period.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

At the end of administration period
Males: Main study group (day 29)
No significant differences were noted in any parameter in the 10 or 100 mg/kg group compared with the control group. In the 1000 mg/kg group, T4 was significantly low and TSH tended to be high, and total cholesterol was
significantly high. γ-GTP, Ca, A/G ratio, albumin, and albumin fraction were significantly high, and T-Bil, β-G and γ-G fractions were significantly low. Among these changes, low T-Bil and high albumin were of low clinical significance and
were not considered toxicologically significant. The high A/G ratio and albumin fraction and low β-G and γ-G fractions were considered corresponding to the high albumin. The high Ca was likely associated with the high albumin and not
toxicologically significant because no changes occurred in IP. The high γ-GTP was not considered toxicologically significant because no changes occurred in AST, ALT, or ALP, and histopathological examination of the liver revealed no changes related to the test article administration other than findings indicative of accelerated metabolism.
Females: Main study group (day 14 after delivery)
No significant differences were noted in any parameter in the 10 mg/kg group compared with the control group. In the 100 mg/kg group, T-Cho was significantly high, which was considered unrelated to the test article administration because no dose-relationship was observed. In the 1000 mg/kg group, T4 was significantly low, and TSH was significantly high. In addition, γ-GTP, Na, A/G ratio, albumin, and albumin fraction were significantly high, and Crea, α1-G and β-G fractions were significantly low in this group. Among these changes, low Crea and high albumin (and associated high A/G ratio and albumin fraction, and low α1-G and β-G fractions) were of low clinical significance and were not considered toxicologically significant. The high Na was likely unrelated to the test article administration because no changes were noted in Cl. The high γ-GTP was not considered toxicologically significant because no changes occurred in AST, ALT, or ALP, histopathological examination of the liver revealed no changes related to the test article administration other than findings indicative of accelerated metabolism, and it was a slight increase. Animal No. 50159 (control group) showed abnormal values of renal parameters including UN, Crea, Ca, and IP, and bilateral yellowish brown discoloration of the kidney, which indicated a decrease in renal function attributable to spontaneous renal lesions. Therefore, data of this animal were considered inappropriate to serve as control data and were excluded from the analyses of blood biochemical parameters except T4 and TSH.

At the end of recovery period
Males: Satellite group (recovery day 15)
In the 1000 mg/kg group, T-Bil was significantly lower and albumin was significantly higher than that in the control group, both of which were of low clinical significance and were not considered toxicologically significant. These
SR16458 changes were noted even at the end of the administration period, and the percentages of T-Bil and albumin in this group to those in the control group were approximately 65.4% and 121.1%, respectively, at the end of the administration period. In contrast, their percentages at the end of the recovery period were approximately 82.8% and 106.6%, respectively, which indicated a tendency toward recovery. In this group, TP, IP, K, and UN were significantly high, which were considered unrelated to the test article administration because individual values were within the ranges of historical control data of the test facility (parameters and means ± 2SDs: TP, 5.05 to 6.17 g/dL; IP, 5.73 to 8.69 mg/dL; K, 4.002 to 4.930 mEq/L; UN, 10.10 to 18.18 mg/dL)2) and these changes were not observed at the end of the administration period.
Females: Satellite group (recovery day 15)
In the 1000 mg/kg group, TSH tended to be high. TSH was also significantly high in the Main study groups (at the end of the administration period); however, significant differences were not noted at the end of the recovery period, which
indicated a tendency toward recovery. Although no significant differences were noted in the means, Crea and UN were high in animal No. 50655; T-Cho was high in animal Nos. 50653 and 50655; and albumin, albumin fraction, and A/G ratio were low, and α1-G fraction was high in animal Nos. 50653, 50654, and 50655. In this group, T-Bil was significantly lower than that in the control group, which was of low clinical significance and was not considered toxicologically significant.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Administration period
Males: Main study group and Satellite group (administration week 4)
No significant differences were noted in any parameter between the 10 or 100 mg/kg group and the control group. In the 1000 mg/kg group, urine volume was significantly high, and urine specific gravity tended to be low compared with the
control group.
Females: Satellite group (administration week 7)
In the 1000 mg/kg group, urine volume was significantly high, and urine specific gravity tended to be low compared with the control group.

Recovery period
Males: Satellite group (recovery week 2)
In the 1000 mg/kg group, urine volume was significantly high, and urine specific gravity was significantly low compared with the control group.
Females: Satellite group (recovery week 2)
In the 1000 mg/kg group, protein tended to be high. No changes were noted in the other parameters.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Administration period
Males: Main study group and Satellite group (administration week 4)
No significant differences were noted in any parameter between the 10 or 100 mg/kg group and the control group. In the 1000 mg/kg group, grip strength of hindlimb was significantly lower than that in the control group, which was
considered an incidental change because individual values of all animals were within the historical control range of the test facility (mean ± 2 SD 313.03 to 888.95 g). No significant differences were noted in the other parameters in this group.
Females: Main study group (lactation day 13)
No significant differences were noted in any parameter between the test article groups and the control group.
Females: Satellite group (administration week 7)
In the 1000 mg/kg group, grip strength of forelimb was significantly higher than that in the control group, which was considered an incidental change because individual values of all animals were within the historical control range of the test
facility (mean ± 2 SD 924.01 to 1569.73 g). No significant differences were noted in the other parameters between the 1000 mg/kg group and the control group.

Recovery period
Males: Satellite group (recovery week 2)
In the 1000 mg/kg group, no significant differences were noted in any parameter compared with the control group.
Females: Satellite group (recovery week 2)
In the 1000 mg/kg group, no significant differences were noted in any parameter compared with the control group.

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):

At the end of administration period
Males: Main study group (day 29)
Histopathological findings in the 100 and 1000 mg/kg groups were as follows: in the liver, centrilobular hypertrophy of hepatocytes of 4 (slight) and 12 (mild) males, in the 100 and 1000 mg/kg groups, respectively; in the kidney, hyaline droplet in proximal tubular epithelium in 12 (slight 3, mild 9) and 11 (slight 1, mild 6, and moderate 4) males, necrosis of renal tubule in 3 (slight) and 10 (slight 8, mild 2) males, cellular cast in 6 (slight 4, mild 2) and 10 (slight 4, mild 3, moderate 3) males, and basophilic change of renal tubule in 12 (slight 11, mild 1) and 12 (slight 3, mild 7, moderate 2) males, in the 100 and 1000 mg/kg groups, respectively. For the hyaline droplet in proximal tubular epithelium of the kidney, immunostaining using anti-α2u-globulin revealed positive reaction; therefore, these changes noted in the kidney were considered attributable to α2u-globulin nephropathy.;in the thyroid, slight hypertrophy of follicular cells in 3 and 10 males, in the 100 and 1000 mg/kg groups, respectively. Among the above changes, frequencies of the followings were significantly higher than those in the control group: hyaline droplet in proximal tubular epithelium, cellular cast, and basophilic change of renal tubule in the kidney in the 100 and 1000 mg/kg groups; and centrilobular hypertrophy of hepatocytes, necrosis of renal tubule in the kidney, and hypertrophy of follicular cells in the thyroid in the 1000 mg/kg group. Other than those described above, various changes were noted, which were considered spontaneous because they were also observed in the control group and no dose-related increase was noted in their frequencies, or because of their low frequencies.
Females: Main study group (day 14 after delivery)
In the 100 and 1000 mg/kg groups, centrilobular hypertrophy of hepatocytes was noted in the liver of 6 (slight) and 12 (mild) females, respectively, and slight hypertrophy of follicular cells was noted in the thyroid in 2 and 4 females,
respectively. In the 1000 mg/kg group, atrophy of the thymus cortex was noted in 8 (slight 5, mild 3) females. Among these changes, frequencies of centrilobular hypertrophy of hepatocytes in the 100 and 1000 mg/kg groups and atrophy of cortex of thymus in the 1000 mg/kg group were significantly higher than those in the control group. In the female with macroscopic finding of dark brown discoloration of the kidney in the 1000 mg/kg group, slight pigmentation of renal tubule was observed, which showed positive reaction to PAS staining and Schmorl staining and was considered to be lipofuscin pigmentation. This change is known to occur spontaneously in rats3), and occurred in only 1 animal; therefore, this was considered unrelated to the test article administration. Other various changes observed were considered spontaneous because they occurred also in the control group, or their frequencies were low.

At the end of recovery period
Males: Satellite group (recovery day 15)
In the 1000 mg/kg group, necrosis of renal tubule was noted in 3 males (slight), cellular cast in 4 males (slight 1, mild 1, moderate 2), and basophilic change of renal tubule in 5 males (slight 1, mild 2, moderate 2). Among them, frequencies of cellular cast and basophilic change of renal tubule were significantly higher than those in the control group. These were noted also at the end of the administration period, and were considered due to α2u-globulin nephropathy. When compared with the results at the end of the administration period, hyaline droplet in proximal tubular epithelium was not found, and the frequency of necrosis of renal tubule tended to decrease. Since cellular cast isindicative of deposition of the cells that was already affected, and basophilic change of renal tubule is a change associated with repair, these changes were considered to be in a recovery phase. Other changes were considered spontaneous because they were also observed in the control group.
Females: Satellite group (recovery day 15)
In the kidney of females in the 1000 mg/kg group, membranous glomerulonephritis was noted in 4 females (slight 1, moderate 3), hyaline cast in 4 females (slight 1, moderate 3), and basophilic change of renal tubule in 4 females (slight 1, mild 3). Frequencies of these finding were significantly higher than those in the control group. Other changes were considered spontaneous because they were also observed in the control group.

The results of the histopathological findings are evaluated as follows,
In the liver, absolute or relative weight was significantly high in males and females in the 100 and 1000 mg/kg groups and necropsy revealed large size and dark brown discoloration in the 1000 mg/kg group at the end of the administration period. However, histopathological examination revealed only centrilobular hypertrophy of hepatocyte and no changes indicating cell damage in these groups, and no changes indicative of hepatic dysfunction were noted in blood biochemistry; therefore, the changes in the liver were considered associated with accelerated drug metabolism and not toxicologically meaningful.

Concerning the thyroid, the relative weight was significantly high in males in the 1000 mg/kg group, and histopathologically hypertrophy of follicular cells was noted in males and females in the 100 and 1000 mg/kg groups at the end of the administration period. In blood biochemistry, T4 was significantly low and TSH tended to be high or was significantly high in males and females in the 1000 mg/kg group at the end of the administration period.
Since T4 is mainly metabolized in the liver by glucuronidation, the metabolism is accelerated by enzyme induction following drug administration, and compensatorily secretion of TSH was increased, which results in hypertrophy of follicular cells in the thyroid4). It has been known that administration of C8 iso-alkane, an analogue of the test article, induces excretion of the metabolites in the urine as glucuronide conjugates, and that C5-C20 alkanes, to which the test article belongs, have similar metabolism5). On that basis, it was considered that glucuronic acid conjugate was increased by enzyme induction following the test article administration, and the changes in the thyroid, T4 and TSH observed in this study were considered indicative of acceleration of drug metabolism in the liver and accompanying physiological response. The low T4 caused by the above mechanism is known to occur commonly in rats owing to the difference in T4-transport protein6), and its associated changes are considered not able to be extrapolated in humans7). Therefore, the changes in the thyroid, T4 and TSH were not considered toxicologically significant. Other than those described above, a significantly low T-Bil was noted with no toxicological significance in males in the 1000 mg/kg group at the end of the administration period, which was considered related to the accelerated metabolism, by a similar mechanism as the low T4. This change also indicated reversibility. T-Bil was significantly low in females only at the end of the recovery period, which was likely due to difference in the physiological conditions of lactating females at the end of the administration period and that of non-mated females at the end of the recovery period.

In the kidney, the absolute and relative weight was high in males in the 100 and 1000 mg/kg groups, and pale discoloration and large size were noted in males in the 1000 mg/kg group in necropsy, and histopathologically α2u-globulin related nephropathy (hyaline droplet in proximal tubular epithelium, necrosis of renal tubule, cellular cast, and basophilic change of renal tubule) was noted in males in the 100 and 1000 mg/kg groups at the end of the administration period. In urinalysis, urine volume was significantly high and specific gravity tended to be low in the 1000 mg/kg group.
These changes were considered related to the findings in the renal tubule. Blood biochemistry revealed high T-Cho, which was slight in degree and no changes were noted in the renal parameters including UN and Crea. It is known that administration of isoparaffins, to which the test article belongs, causes α2u-globulin nephropathy in male rats 5,8), and the changes in the kidney and associated change in urinalysis and blood chemistry noted in this study were also considered to be effects of the test article administration. α2u-globulin nephropathy is specific to male rats and is not able to be extrapolated in humans9), therefore, these changes were not considered toxicologically significant. At the end of recovery period, urine volume still was significantly high and specific gravity was significantly low; while reversibility was shown for T-Cho, weight, and macroscopic and histopathological findings of the kidney.
Females in the 1000 mg/kg group showed increasing tendency of absolute and relative weight of the kidney, and large size, pale discoloration, and rough surface of the kidney, and histopathologically membranous glomerulonephritis, hyaline cast, and basophilic change of renal tubule at the end of the recovery period. These findings were considered related to the test article administration. In this group, urine volume was significantly high and specific gravity tended to be low during the administration period, which were considered related to the renal findings. The increasing tendency of protein in urinalysis in the recovery period and high UN, Crea, and T-Cho and low albumin and albumin fraction in blood biochemistry at the end of the recovery period were considered associated with glomerular change. The high α1-G fraction was considered due to an increase in the protein that is a component of α1-G fraction because low TP values were not noted, and the low A/G ratio was considered attributable to the changes of albumin fraction and α1-G fraction. Similar changes were not observed in the Main study groups. This was likely because females at the end of the recovery period were non-mated, and their physiological conditions were different from those of lactating females in the Main study groups.

Concerning the thymus, significantly low absolute and relative weight and small size of the thymus, and histopathologically atrophy of the thymus cortex were noted in females in the 1000 mg/kg group at the end of the administration period.
These were considered to be changes secondary to the low body weight, or caused by stress because no effects of the test article administration were noted in the other immune tissues such as the lymphoid tissues and the spleen. In the uterus, the absolute and relative weight was low, which was considered to be a debilitating change because no abnormalities were noted histopathologically in females in the 1000 mg/kg group at the end of the administration period. These changes indicated reversibility.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

At the end of administration period
Males: Main study group (day 29)
Histopathological findings in the 100 and 1000 mg/kg groups were as follows: in the liver, centrilobular hypertrophy of hepatocytes of 4 (slight) and 12 (mild) males, in the 100 and 1000 mg/kg groups, respectively; in the kidney, hyaline droplet in proximal tubular epithelium in 12 (slight 3, mild 9) and 11 (slight 1, mild 6, and moderate 4) males, necrosis of renal tubule in 3 (slight) and 10 (slight 8, mild 2) males, cellular cast in 6 (slight 4, mild 2) and 10 (slight 4, mild 3, moderate 3) males, and basophilic change of renal tubule in 12 (slight 11, mild 1) and 12 (slight 3, mild 7, moderate 2) males, in the 100 and 1000 mg/kg groups, respectively. For the hyaline droplet in proximal tubular epithelium of the kidney, immunostaining using anti-α2u-globulin revealed positive reaction; therefore, these changes noted in the kidney were considered attributable to α2u-globulin nephropathy.;in the thyroid, slight hypertrophy of follicular cells in 3 and 10 males, in the 100 and 1000 mg/kg groups, respectively. Among the above changes, frequencies of the followings were significantly higher than those in the control group: hyaline droplet in proximal tubular epithelium, cellular cast, and basophilic change of renal tubule in the kidney in the 100 and 1000 mg/kg groups; and centrilobular hypertrophy of hepatocytes, necrosis of renal tubule in the kidney, and hypertrophy of follicular cells in the thyroid in the 1000 mg/kg group. Other than those described above, various changes were noted, which were considered spontaneous because they were also observed in the control group and no dose-related increase was noted in their frequencies, or because of their low frequencies.
Females: Main study group (day 14 after delivery)
In the 100 and 1000 mg/kg groups, centrilobular hypertrophy of hepatocytes was noted in the liver of 6 (slight) and 12 (mild) females, respectively, and slight hypertrophy of follicular cells was noted in the thyroid in 2 and 4 females,
respectively. In the 1000 mg/kg group, atrophy of the thymus cortex was noted in 8 (slight 5, mild 3) females. Among these changes, frequencies of centrilobular hypertrophy of hepatocytes in the 100 and 1000 mg/kg groups and atrophy of cortex of thymus in the 1000 mg/kg group were significantly higher than those in the control group. In the female with macroscopic finding of dark brown discoloration of the kidney in the 1000 mg/kg group, slight pigmentation of renal tubule was observed, which showed positive reaction to PAS staining and Schmorl staining and was considered to be lipofuscin pigmentation. This change is known to occur spontaneously in rats3), and occurred in only 1 animal; therefore, this was considered unrelated to the test article administration. Other various changes observed were considered spontaneous because they occurred also in the control group, or their frequencies were low.

At the end of recovery period
Males: Satellite group (recovery day 15)
In the 1000 mg/kg group, necrosis of renal tubule was noted in 3 males (slight), cellular cast in 4 males (slight 1, mild 1, moderate 2), and basophilic change of renal tubule in 5 males (slight 1, mild 2, moderate 2). Among them, frequencies of cellular cast and basophilic change of renal tubule were significantly higher than those in the control group. These were noted also at the end of the administration period, and were considered due to α2u-globulin nephropathy. When compared with the results at the end of the administration period, hyaline droplet in proximal tubular epithelium was not found, and the frequency of necrosis of renal tubule tended to decrease. Since cellular cast isindicative of deposition of the cells that was already affected, and basophilic change of renal tubule is a change associated with repair, these changes were considered to be in a recovery phase. Other changes were considered spontaneous because they were also observed in the control group.
Females: Satellite group (recovery day 15)
In the kidney of females in the 1000 mg/kg group, membranous glomerulonephritis was noted in 4 females (slight 1, moderate 3), hyaline cast in 4 females (slight 1, moderate 3), and basophilic change of renal tubule in 4 females (slight 1, mild 3). Frequencies of these finding were significantly higher than those in the control group. Other changes were considered spontaneous because they were also observed in the control group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

The results of the histopathological findings are evaluated as follows:
In the liver, absolute or relative weight was significantly high in males and females in the 100 and 1000 mg/kg groups and necropsy revealed large size and dark brown discoloration in the 1000 mg/kg group at the end of the administration period. However, histopathological examination revealed only centrilobular hypertrophy of hepatocyte and no changes indicating cell damage in these groups, and no changes indicative of hepatic dysfunction were noted in blood biochemistry; therefore, the changes in the liver were considered associated with accelerated drug metabolism and not toxicologically meaningful.

Concerning the thyroid, the relative weight was significantly high in males in the 1000 mg/kg group, and histopathologically hypertrophy of follicular cells was noted in males and females in the 100 and 1000 mg/kg groups at the end of the administration period. In blood biochemistry, T4 was significantly low and TSH tended to be high or was significantly high in males and females in the 1000 mg/kg group at the end of the administration period.
Since T4 is mainly metabolized in the liver by glucuronidation, the metabolism is accelerated by enzyme induction following drug administration, and compensatorily secretion of TSH was increased, which results in hypertrophy of follicular cells in the thyroid4). It has been known that administration of C8 iso-alkane, an analogue of the test article, induces excretion of the metabolites in the urine as glucuronide conjugates, and that C5-C20 alkanes, to which the test article belongs, have similar metabolism5). On that basis, it was considered that glucuronic acid conjugate was increased by enzyme induction following the test article administration, and the changes in the thyroid, T4 and TSH observed in this study were considered indicative of acceleration of drug metabolism in the liver and accompanying physiological response. The low T4 caused by the above mechanism is known to occur commonly in rats owing to the difference in T4-transport protein6), and its associated changes are considered not able to be extrapolated in humans7). Therefore, the changes in the thyroid, T4 and TSH were not considered toxicologically significant. Other than those described above, a significantly low T-Bil was noted with no toxicological significance in males in the 1000 mg/kg group at the end of the administration period, which was considered related to the accelerated metabolism, by a similar mechanism as the low T4. This change also indicated reversibility. T-Bil was significantly low in females only at the end of the recovery period, which was likely due to difference in the physiological conditions of lactating females at the end of the administration period and that of non-mated females at the end of the recovery period.

In the kidney, the absolute and relative weight was high in males in the 100 and 1000 mg/kg groups, and pale discoloration and large size were noted in males in the 1000 mg/kg group in necropsy, and histopathologically α2u-globulin related nephropathy (hyaline droplet in proximal tubular epithelium, necrosis of renal tubule, cellular cast, and basophilic change of renal tubule) was noted in males in the 100 and 1000 mg/kg groups at the end of the administration period. In urinalysis, urine volume was significantly high and specific gravity tended to be low in the 1000 mg/kg group.
These changes were considered related to the findings in the renal tubule. Blood biochemistry revealed high T-Cho, which was slight in degree and no changes were noted in the renal parameters including UN and Crea. It is known that administration of isoparaffins, to which the test article belongs, causes α2u-globulin nephropathy in male rats 5,8), and the changes in the kidney and associated change in urinalysis and blood chemistry noted in this study were also considered to be effects of the test article administration. α2u-globulin nephropathy is specific to male rats and is not able to be extrapolated in humans9), therefore, these changes were not considered toxicologically significant. At the end of recovery period, urine volume still was significantly high and specific gravity was significantly low; while reversibility was shown for T-Cho, weight, and macroscopic and histopathological findings of the kidney.
Females in the 1000 mg/kg group showed increasing tendency of absolute and relative weight of the kidney, and large size, pale discoloration, and rough surface of the kidney, and histopathologically membranous glomerulonephritis, hyaline cast, and basophilic change of renal tubule at the end of the recovery period. These findings were considered related to the test article administration. In this group, urine volume was significantly high and specific gravity tended to be low during the administration period, which were considered related to the renal findings. The increasing tendency of protein in urinalysis in the recovery period and high UN, Crea, and T-Cho and low albumin and albumin fraction in blood biochemistry at the end of the recovery period were considered associated with glomerular change. The high α1-G fraction was considered due to an increase in the protein that is a component of α1-G fraction because low TP values were not noted, and the low A/G ratio was considered attributable to the changes of albumin fraction and α1-G fraction. Similar changes were not observed in the Main study groups. This was likely because females at the end of the recovery period were non-mated, and their physiological conditions were different from those of lactating females in the Main study groups.

Concerning the thymus, significantly low absolute and relative weight and small size of the thymus, and histopathologically atrophy of the thymus cortex were noted in females in the 1000 mg/kg group at the end of the administration period.
These were considered to be changes secondary to the low body weight, or caused by stress because no effects of the test article administration were noted in the other immune tissues such as the lymphoid tissues and the spleen. In the uterus, the absolute and relative weight was low, which was considered to be a debilitating change because no abnormalities were noted histopathologically in females in the 1000 mg/kg group at the end of the administration period. These changes indicated reversibility.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

2

Key result

Dose descriptor:

NOAEL

Remarks:

reproduction

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse and treatment-related effects were observed at 1000 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Death or missing (possibly cannibalized by maternal rats) was sporadically observed in all test article groups from postnatal days 0 to 13. However, these occurred at low incidence and no significant difference was noted in the viability index between the test article groups and the control group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, body weight was significantly low in males and females on postnatal days 0 to 13.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

the serum T4 concentrations were examined, no abnormal changes were noted.

Urinalysis findings:

not examined

Sexual maturation:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, anogenital distance divided by the cube root of body weight was significantly high in males; this change was not considered toxicologically significant because of being high values.

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In dead offspring, dilatation of renal pelvis was noted in 2 offspring from different litters in the 1000 mg/kg group. This was observed in only 2 offspring; therefore it was considered unrelated to the test article administration.

Histopathological findings:

not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Overall reproductive toxicity

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day

Treatment related:

yes

Relation to other toxic effects:

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

Dose response relationship:

no

Relevant for humans:

not specified

Any other information on results incl. tables

Table 1

Estrous cycles

Generation: F0 Species: Rat

Test article Dose		/Before mating
		Mean length of estrous cycle (Days)	Number of estrus	Number of animals with acyclic or irregular cycle
IP solvent 1620 0 mg/kg	n	12	12	12
	Mean	4.04	3.5	(0)
	S.D.	0.14	0.5
IP solvent 1620 10 mg/kg	n	12	12	12
	Mean	4.00	3.4	(0)
	S.D.	0.00	0.5
IP solvent 1620 100 mg/kg	n	12		12
	Mean	4.10	3.5	(1)
	S.D.	0.33	0.5
IP solvent 1620 1000 mg/kg	n	12	12	12
	Mean	4.46	3.5	(2)
	S.D.	0.89	0.7

() : Values in brackets represent number of animals with acyclic or irregular cycle.

Not significantly different from IP solvent 1620 0 mg/kg

 

Table 2 - 1

Reproductive performance

Generation: F0 Sex: Male Species: Rat

1st mating

2nd mating

Total

Test article Dose

Number of pairs

Day of conceiving

Copulation index (%)

Fertility index (%)

Copulation index (%)

Fertility index (%)

Copulation index (%)

Fertility index (%)

IP solvent 1620 0 mg/kg

n

12

12

(12/12)

(12/12)

(12/12)

(12/12)

Mean

2.8

100.0

100.0

100.0

100.0

S.D.

1.2

IP solvent 1620 10 mg/kg

n

12

12

(12/12)

(12/12)

(12/12)

(12/12)

Mean

2.5

100.0

100.0

100.0

100.0

S.D.

0.7

EF

EF

IP solvent 1620 100 mg/kg

n

12

12

(12/12)

(12/12)

(12/12)

(12/12)

Mean

2.8

100.0

100.0

100.0

100.0

S.D.

1.2

EF

EF

IP solvent 1620 1000 mg/kg

n

12

12

(12/12)

(12/12)

(12/12)

(12/12)

Mean

2.8

100.0

100.0

100.0

100.0

S.D.

0.9

EF

EF

Not significantly different from IP solvent 1620 0 mg/kg

EF : The averages of all groups are same and all data is 0 in frequency. (all data of all groups is identical.)

 

Table 2 - 2

Reproductive performance

Generation : F0 Sex : Female Species : Rat

				1st mating		2nd mating		Total
Test article Dose		Number of pairs	Day of conceiving	Copulation index (%)	Fertility index (%)	Copulation index (%)	Fertility index (%)	Copulation index (%)	Fertility index (%)
IP solvent 1620 0 mg/kg	n	12	12	(12/12)	(12/12)			(12/12)	(12/12)
	Mean		2.8	100.0	100.0			100.0	100.0
	S.D.		1.2
IP solvent 1620 10 mg/kg	n	12	12	(12/12)	(12/12)			(12/12)	(12/12)
	Mean		2.5	100.0	100.0			100.0	100.0
	S.D.		0.7
								EF	EF
IP solvent 1620 100 mg/kg	n	12	12	(12/12)	(12/12)			(12/12)	(12/12)
	Mean		2.8	100.0	100.0			100.0	100.0
	S.D.		1.2
								EF	EF
IP solvent 1620 1000 mg/kg	n	12	12	(12/12)	(12/12)			(12/12)	(12/12)
	Mean		2.8	100.0	100.0			100.0	100.0
	S.D.		0.9
								EF	EF

 

Table 3 – 1

Delivery data

Generation : F0 Species : Rat

		Gestation period	Number of implantation	Birth index	Number of offspring	Number of live newborns			Sex rate (offspring)	Number of dead newborns			Gestation index
Test article Dose
		(day)		(%)		M	F	Total	(%)	Dead	Cannibalism	Total	(%)
IP solvent 1620 0 mg/kg	n	12	12	12	12	12	12	12	67/148	12	12	12	12/12
	Mean	22.25	13.3	93.71	12.3	5.6	6.8	12.3	45.3	0.0	0.0	0.0	100.0
	S.D.	0.45	4.1	9.95	3.7	2.0	2.6	3.7		0.0	0.0	0.0	0.00
IP solvent 1620 10 mg/kg	n	12	12	12	12	12	12	12	91/176	12	12	12	12/12
	Mean	22.33	15.7	93.34	14.8	7.6	7.1	14.7	51.7	0.1	0.0	0.1	100.0
	S.D.	0.49	1.4	8.24	2.1	2.6	2.5	2.2		0.3	0.0	0.3	0.00
													EF
IP solvent 1620 100 mg/kg	n	12	12	12	12	12	12	12	80/163	12	12	12	12/12
	Mean	22.25	15.2	88.74	14.0	6.7	6.9	13.6	49.1	0.3	0.2	0.4	100.0
	S.D.	0.45	2.1	13.76	2.6	3.1	2.9	3.3		0.9	0.6	1.4	0.00
													EF
IP solvent 1620 1000 mg/kg	n	12	12	12	12	12	12	12	81/161	12	12	12	12/12
	Mean	22.25	15.2	88.26	13.8	6.8	6.7	13.4	50.3	0.3	0.0	0.3	100.0
	S.D.	0.45	1.7	12.22	2.8	1.7	1.7	2.6		0.7	0.0	0.7	0.00
													EF

M: Male, F: Female

Not significantly different from IP solvent 1620 0 mg/kg

EF: The averages of all groups are same and all data is 0 in frequency. (all data of all groups is identical.)

 

Table 3 – 2

Delivery data

External examination of offspring Days after birth : 0

Generation : F0 Species : Rat

Test article Dose Dose unit Number of dams Number of offspring	IP solvent 1620 0 mg/kg 12 148		IP solvent 1620 10mg/kg 12 176	IP solvent 1620 100 mg/kg 12 163	IP solvent 1620 1000 mg/kg 12 161
Number of dams with anomalous offspring (incidence %)		0(0.00)	0(0.00)	1(8.33)	0(0.00)
Number of offspring with any anomaly (incidence %)		0(0.00)	0(0.00)	1(0.59)	0(0.00)
Absent tail	n	0(0)	0(0)	1(1)	0(0)
	Mean			0.59
	S.D.			2.05

 

Table 4

Clinical signs of offspring

Generation: F0 Species : Rat

Test article Dose	Clinical signs		/Before culling					/After culling
		Days after birth	0	1	2	3	4	5	6	7	8	9	10	11	12	13
IP solvent 1620 0 mg/kg	Number of dams examined		12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Number of offspring		148	148	148	148	148	91	91	91	91	91	91	91	91	91
	Number of dams with anomalous offspring		0	0	0	0	0	0	0	0	0	0	0	0	0	0
IP solvent 1620 10 mg/kg	Number of dams examined		12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Number of offspring		176	176	176	176	173	96	96	96	96	96	96	96	96	96
	Number of dams with anomalous offspring		0	0	0	0	0	0	0	0	0	0	0	0	0	0
IP solvent 1620 100 mg/kg	Number of dams examined		12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Number of offspring		163	163	163	163	161	95	95	95	95	95	95	95	95	95
	Number of dams with anomalous offspring		0	0	0	0	0	0	0	0	0	0	0	0	0	0
IP solvent 1620 1000 mg/kg	Number of dams examined		12	12	12	12	12	12	12	12	12	12	12	12	12	12
	Number of offspring		161	157	157	157	154	95	95	95	95	95	93	93	93	93
	Number of dams with anomalous offspring		0	0	0	0	0	0	0	0	0	0	0	0	0	0

 

Table 5

Litter sizes and viability indices of offspring

Generation : F0 Species : Rat

	/Days after birth 0		0	4	4	4	13	13
Test article Dose			Viability index (%)	Pre-culled	Viability index (%)	Post-culled		Viability index (%)
IP solvent 1620 0 mg/kg	Total	148		148		91	91
	n	12	12	12	12	12	12	12
	Mean		100.00		100.00			100.00
	S.D.		0.00		0.00			0.00
IP solvent 1620 10 mg/kg	Total	176		173		96	96
	n	12	12	12	12	12	12	12
	Mean		99.31		98.46			100.00
	S.D.		2.40		2.80			0.00
IP solvent 1620 100 mg/kg	Total	163		161		95	95
	n	12	12	12	12	12	12	12
	Mean		96.53		98.92			100.00
	S.D.		12.04		2.54			0.00
IP solvent 1620 1000 mg/kg	Total	161		154		96	93
	n	12	12	12	12	12	12	12
	Mean		97.83		95.66			96.88
	S.D.		4.29		7.62			5.65

 

Table 6

Number of papillae in male offspring  Period: F1 postnatal Day 13

Sex: Male Species : Rat

Test article Dose		Number of papillae
IP solvent 1620 0 mg/kg	n	12
	Mean	0.00
	S.D.	0.00
IP solvent 1620 10 mg/kg	n	12
	Mean	0.00
	S.D.	0.00
IP solvent 1620 100 mg/kg	n	12
	Mean	0.00
	S.D.	0.00
IP solvent 1620 1000 mg/kg	n	12
	Mean	0.00
	S.D.	0.00

 

Table 7

Body weights of offspring

Generation: F0 Unit : g Species : Rat

Test article Dose			/Days after birth
			0	4	7	13
IP solvent 1620 0 mg/kg	Male	n	12	12	12	12
		Mean	6.953	11.385	19.110	35.599
		S.D.	0.593	1.187	1.588	2.388
	Female	n	12	12	12	12
		Mean	6.599	10.874	18.333	34.686
		S.D.	0.663	1.388	1.333	2.309
IP solvent 1620 10 mg/kg	Male	n	12	12	12	12
		Mean	6.889	10.808	19.179	35.644
		S.D.	0.606	1.360	1.902	3.415
	Female	n	12	12	12	12
		Mean	6.478	10.372	18.159	34.234
		S.D.	0.512	1.423	1.812	2.864
IP solvent 1620 100 mg/kg	Male	n	12	12	12	12
		Mean	6.681	11.400	19.145	36.559
		S.D.	0.712	1.842	2.648	3.630
	Female	n	12	12	12	12
		Mean	6.322	10.719	18.233	35.142
		S.D.	0.795	1.645	2.376	3.303
IP solvent 1620 1000 mg/kg	Male	n	12	12	12	12
		Mean	5.939	8.878	14.723	26.938
		S.D.	0.478 DT 	1.291 DT 	1.409 DT 	2.108 DT 
	Female	n	12	12	12	12
		Mean	5.648	8.612	14.097	26.158
		S.D.	0.455 DT 	1.173 DT 	1.577 DT 	2.377 DT 

 

Table 8

Anogenital distance of offspring  Period: F1 postnatal Day 4

Sex: Male, Female  Species: Rat

Test article Dose		Male		Female
		AGD (mm)	AGD/(BW)1/3	AGD (mm)	AGD/(BW)1/3
IP solvent 1620 0 mg/kg	n	12	12	12	12
	Mean	5.695	2.536	2.809	1.272
	S.D.	0.481	0.176	0.273	0.098
IP solvent 1620 10 mg/kg	n	12	12	12	12
	Mean	5.683	2.576	2.639	1.212
	S.D.	0.374	0.100	0.234	0.074
IP solvent 1620 100 mg/kg	n	12	12	12	12
	Mean	5.721	2.549	2.710	1.233
	S.D.	0.337	0.105	0.209	0.050
IP solvent 1620 1000 mg/kg	n	12	12	12	12
	Mean	5.582	2.708	2.626	1.286
	S.D.	0.378	0.128 DT**	0.208	0.092

 

Table 9 – 1

Serum T4 concentration of offspring  Stage: F1 postnatal Day 4

Sex: Male, Female  Species : Rat

Test article Dose		T4 ng/mL
IP solvent 1620 0 mg/kg	n	10
	Mean	14.725
	S.D.	3.560
IP solvent 1620 10 mg/kg	n	12
	Mean	20.472
	S.D.	7.816
IP solvent 1620 100 mg/kg	n	10
	Mean	17.591
	S.D.	3.447
IP solvent 1620 1000 mg/kg	n	12
	Mean	19.258
	S.D.	5.688

 

Table 9– 2

Serum T4 concentration of offspring  Stage : F1 postnatal Day 13

Sex: Male, Female  Species : Rat

Test article Dose		T4 ng/mL
IP solvent 1620 0 mg/kg	n	12
	Mean	30.430
	S.D.	5.360
IP solvent 1620 10 mg/kg	n	12
	Mean	30.741
	S.D.	6.433
IP solvent 1620 100 mg/kg	n	12
	Mean	29.947
	S.D.	4.730
IP solvent 1620 1000 mg/kg	n	12
	Mean	31.302
	S.D.	3.599

 

Table 10

Necropsy findings of offspring

Generation : F0  Species : Rat

Test article	IP solvent 1620	IP solvent 1620	IP solvent 1620	IP solvent 1620
Dose	0	10	100	1000
Dose unit	mg/kg	mg/kg	mg/kg	mg/kg
Number of dams	12	12	12	12
Number of live offspring examined on Day 13 after birth	91	96	95	93
Finding absent	91	96	95	93
Number of dead offspring examined on Day 0-13 after birth	0	2	6	7
Finding absent	0	2	6	5
Dilatation, renal pelvis	0	0	0	2

 

Applicant's summary and conclusion

Conclusions:

The NOAEL of test substance was considered to be 100 mg/kg bw/day for general systemic toxicity of both male and female parental animals;
The NOAEL of test substance was considered to be 1000 mg/kg bw/day for reproductive performance of both male and female parental animals;
The NOAEL of test substance was considered to be 100 mg/kg bw/day for development of offspring.