Carboxylic acids, C5-9

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Octanoic acid

EC Number:

204-677-5

EC Name:

Octanoic acid

Cas Number:

124-07-2

Molecular formula:

C8H16O2

IUPAC Name:

octanoic acid

Test material form:

liquid

Specific details on test material used for the study:

Test substance octanoic acid, alternate name: n-caproic acid; English chemical name: octanoic acid, CAS No. 124-07-2, Ministerial Circular Publication Adjustment Number (Chemical Evaluation Act) :2-608, Fig.1]; chemical structure:CH3(CH2)6COOH, molecular weight: 144.21, physical properties, characteristics, extremely faint yellow clear liquid with distinctive odor; does not dissolve in water, readily dissolves in ethanol and acetone; ignition point: 109°C; density (20°C): 0.910g/mL

Test animals

Species:

rat

Strain:

other: Crl:CD (SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The types of animals used in this study were generally those used in toxicity tests and we used CrF(SD) female and male rats (SPF) on a clear systematic basis. We obtained 62 male and 42 female animals aged 7 weeks in the main test group on October 20, 2010 and 62 female parents aged 7 weeks as the mating group. The body weight range one 1 after we obtained them was 230 to 250 g for the males in the main test group, 165 to 193 g for the females in the main test group and 155 to 181 g for the female parents in the mating group.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.5w/v% methyl cellulose solution

Details on exposure:

The test dose was 62.5, 250 and 1000 mg/kg/day. In the main test group, the dose was administered for 28 days, and a recovery period of 14 days was set. In the mating group, the dose was administered for 42 to 46 days: up to 14 days before mating, during the mating period, during the gestation period and day 4 of lactation.

Details on mating procedure:

We bred separate male and female groups each consisting of 2 animals per single cage using a stainless steel suspension type cage (W: 240 x D: 380 X H: 200mm) during the quarantine and acclimation period. After we separated them into groups, we bred them individually using a stainless steel suspension type cage. Mating was carried out inside the cage. On day 18 of gestation, we transferred each female parent in the group to a plastic cage (W: 310 x D: 360 x H:175mm) provided with animal bedding which had been treated with an autoclave and allowed the parent females to give birth naturally and to lactate them to give birth naturally and to lactate. On day 4 of lactation, we bred the parent females in the mating group individually using a stainless steel suspension type cage. We changed the water bottle, the feeding saucer on the stainless steel type cage and the plastic cage at least twice a week and changed the stainless steel suspension type cage and the feed dish at least once every 2 weeks. We cleaned the animal breeding room (floor was swept) and floor was mopped with 0.02 % sodium hypochloride acqueous solution and disinfected it once a day.
We carried out microbiological monitoring tests from the monitored animals after the necropsy of the mating group female parents was terminated (Mucoplasma spp., Clostindium piliforme, HVJ, MHV, Corvnebactenum kuscheri and SDAV). No aberrations were seen which could suggest infection in the microbiological monitoring test carried out at Kitayama Rabes Ltd.

Duration of treatment / exposure:

28 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

250 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:

There were 12 each males and females in the control group and the 62.5, 250 and 1000 mg/kg groups.

Control animals:

yes, concurrent vehicle

Examinations

Parental animals: Observations and examinations:

We checked for any deaths and observed the group’s general condition twice a day pre-administration and once post-administration (1-120 minutes after administration), and once on the day of necropsy during the administration period. The group’s condition was kept under observation.

Postmortem examinations (parental animals):

We measured the body weight of the female parents that did not give birth by 10 AM of day 25 of gestation (electronic scales: PB3002, Metrotred Ltd). Then, we exsanguinated the animals from the abdominal aorta under anesthesia by abdominal administration of sodium pentobarbital (40 mg/kg), euthanized them, carried out necropsies and confirmed gestation.
Female parents with no confirmed nidation were considered non-gestational.
We fixed the spleen, sublingual gland, submandibular gland, ovaries, uterus, vagina and mammary glands in 10 vol% neutral buffer formalin and stored them.
The body weight of the parental females was a reference value and was created only for the individual tables.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Mortality:

no mortality observed

Description (incidence):

Males During Administration Period:
There were no cases of deaths or moribund animals in any of the groups. After administration in the 1000 mg/kg group, there were 10 cases of transitory salivation. No aberrations in the general condition were noted in the 250 and 62.5 mg/kg group or in the control groups.

Females During Administration Period:
There were no cases of death or moribund animals in any of the groups. After administration in the 100 mg/kg group, there were 4 cases of transitory salivation in the 1000 mg/kg group. No aberrations in the general condition were noted in the 250 and 62.6 mg/g group or in the control groups.

Males During Recovery Period:
There were no cases of deaths or moribund animals in any of the groups.
No aberrations were seen in the general condition in any of the groups.

Females During Recovery Period:
There were no cases of deaths or moribund animals in any of the groups. No aberrations in the general condition were noted in the 1000 mg/kg group and in the control groups.

Mating Group Female Parents:
There were no cases of deaths, moribund animals, salivation or premature births in any of the groups. There were 6 cases of transitory salivation in the 1000 mg/kg group before mating started and during the mating period and 3 cases during the gestation period. There were no aberrations in the general conditions in the 250 and 62.5 mg/kg group and in the control groups before mating started, during the mating period, during the gestation period and during the lactation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Males During Administration Period:
No significant difference was seen in the body weight on each measuring day in all of the administration groups compared to the control groups.

Females During Administration Period:
There were no significant differences in body weight on each measuring day for all of the administration groups compared to the control groups.

Males During Recovery Period:
There were no significant differences in body weight for all of the administration groups compared to the control groups.

Females During Recovery Period:
There were no significant differences body weight on each measuring day for the 1000 mg/kg group compared to the control group.

Mating Group Female Parents:
There were no significant differences in body weight on each measuring day for all of the administration groups compared to the control groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Males During Administration Period:
There were no significant differences in the amount ingested on each measuring day for all of the administration groups compared to the control groups.

Females During Administration Period:
No significant high values were seen in the 62.5 mg/kg group compared to the control groups, however, there were no changes which were dose dependent. As a result, there were no significant differences in the amount ingested on each measuring day in the 1000 and 250 mg/kg groups which could be thought to be toxicologically influential as compared to the control groups.

Males During Recovery Period:
There were no significantly high values in the amount ingested on day 2 of recovery in the 62.5mg/kg group compared to the control groups, however, there were no dose-dependent changes so that there were no significant differences in the amount ingested on each measuring day in the 1000 and 250 mg/kg group which could be thought to be toxicologically influential as compared to the control groups.

Females During Recovery Period:
The 1000mg / kg group showed a significantly lower food intake on the 5th day of recovery compared to the control group.
No abnormalities were observed on other measurement days, and this was not considered a toxic biological effect.

Mating Group Female Parents:
No significant differences were seen in the amount ingested on each measuring day for all of the administration groups compared to the control groups.

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

no effects observed

Description (incidence and severity):

Males During Administration Period:
No significant differences were seen in the amount of water ingested on each measuring day for all of the administration groups compared to the control groups.

Females During Administration Period:
No significant differences were seen in the amount of water ingested on each measuring day for all of the administration groups compared to the control groups.
Males During Recovery Period:
No significant differences were seen in the amount of water ingested on each measuring day in all of the administration groups compared to the control groups.
Females During Recovery Period:
No significant differences were seen in the amount of water ingested on each measuring day in the 1000 mg/kg group compared to the control groups.

Mating Group Female Parents:
No significantly high values were seen in the amount of water ingested on administration day 9 in the 1000 mg/kg group compared to the control groups, however, as no aberrations were noted in the main test group, there were no significant differences in the amount of water ingested on each measuring day in the 250 and 62.5 mg/kg group compared to the control group which could be thought to have a toxicological effect.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males When Administration Period Terminated:
No significant difference was seen in any of the measuring items for any of the administration groups
compared to the control groups.
Females When Administration Period Terminated:
No significant difference was seen in any of the measuring items for any of the administration groups
compared to the control groups.
Males When Recovery Period Terminated:
No significant difference was seen in any of the measuring items in any of the administration groups
compared to the control groups.
Female When Recovery Period Terminated:
The 1000 mg / kg group showed significantly higher MCHC than the control group, but because the
difference from the control group was slight, the red blood cell count and hemoglobin level were
unchanged, and because it was within the range of background data of the laboratory [MCHC: 36.1
Sat 0.5 (g / dL); Attachment 11] this was not thought to be affected by the test substance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Males When Administration Period Terminated:
No significantly low values were seen in the urea nitrogen and no significantly high values were see
n in the inorganic phosphorus in the1000 mg/kg group compared to the control group. No significant
difference was seen in any of the measuring items in the 250 and 62.5 mg/kg groups compared to
the control groups.
Females When Administration Period Terminated:
No significantly high potassium values were seen in the 1000 mg/kg group compared to the control
groups. No significant difference was seen in any of the measuring items in the 250 and 62.5 mg/kg
groups compared to the control groups.
Males When Recovery Period Terminated:
No significant difference was seen in any of the measuring items in any of the administration groups
compared to the control groups.
Females When Recovery Period Terminated:
No significant difference was noted in any of the measuring items in the 1000 mg/kg group compared
to the control groups.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Males When Administration Period Terminated:
There were no significant differences in urine volume or urine specific gravity in each treatment group
compared to the control group.
The color tone, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen and sediment were
approximately the same for the control groups.
Females When Administration Group Terminated:
No significant difference was seen in the amount of urine and the urine specific weight for any of th
e administration groups compared to the control groups. The color tone, pH, protein, glucose, keton
es, bilirubin, occult blood, urobilinogen and sediment were approximately the same for all of the a
dministration groups.
Males When Recovery Period Terminated:
No significantly high values were seen in the urine specific weight in the 1000 mg/kg group compared
to the control groups.
The color tone, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen and sediment were
approximately the each for all of the administration groups compared to the control groups.
Female When Recovery Period Terminated:
No significant differences were seen in the amount of urine and the urine specific weight in the 1000
mg/kg group compared to the control groups.
The color tone, pH, protein, glucose, ketones, bilirubin, occult blood, urobilinogen and sediment were
approximately the same in the 1000 mg/kg compared to the control groups.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Males When Administration Period Terminated:
There were no significantly low values in the number of times of general standing up measured at 10
day intervals in the 62.5 mg/kg group compared to the control groups, however, there were no dose d
ependent changes so that these were not thought to be toxicologically influential.
Females When Administration Period Terminated:
There were no significant differences in any of the items for any of the administration groups
compared to the control groups.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Males When Administration Period Terminated:
Lungs: one-sided foreign matter granuloma was seen in 1 case. Stomach：hyperplasia of the anterior
stomach squamous epithelium was seen in 6 case in the 1000 and 250 mg/kg groups and in 2 cases
in the 62.5 mg/kg group. The degree was moderate in the 1000 mg/kg group, mild in the 250 mg/kg
group and extremely mild in the 62. 5 mg/kg group. In addition, stomach ulcers were seen in 3 cases
in the 250 mg/kg group and the degree was extremely mild. Of these findings, a significant difference
in the hyperplasia of the anterior stomach squamous was confirmed in the 1000 and 250 mg/kg gro
ups compared to the control groups and a dose responsiveness was confirmed.
Other changes were obtained in the following findings.
Pancreas: lobule atrophy was seen in 1 case in the control group.
Testes: there was 1 case of double-side atrophy of the seminiferous tubule at time of necropsy, how
ever, this was a case in which there was a diminution and softening of the testes and a diminution of
the epididymis.
Epididymis: there was 1 case of double-sided atrophy and cellular residue inside the lumen, however,
this was in the control group. However, this was a case of diminution and softening of the testes and
diminution of the epididymis during the necropsy.
Prostate gland ventral lobe: there was 1 case of cell infiltration in the control group and 2 cases in the
1000 mg/kg group. The 1 case in the control group was a case in which there was a diminution and a
softening of the testes and a diminution of the epididymis during necropsy.
Pituitary gland: there was 1 case of a cyst in the 1000 mg/kg group.
Ophthalmus: there was 1 case of one-sided localized retinal atrophy in the 1000 mg/kg group.
Furthermore, the changes seen in the 1000 mg/kg group were findings which were usually observe
d in the control groups. There was no difference in the incidence with the control group and it was
determined to be an incidental change. In addition, there were no aberrations seen in the heart,
trachea, sublingual gland, submandibular gland, esophagus, duodenum, colon, ileum (including
Peyer’s patch), the appendix, the intestine, the rectum, the thymus, the spleen, the submandibular
lymph nodes, the mesenteric lymph nodes
the kidneys, the scrotal sac (including the hard gland), the adrenal gland, the thyroid (including the
parathyroid gland), the cerebrum, the cerebellum, the pons, the spinal cord, the sciatic nerve, the
Harderian gland, the sternum (including the medulla ossium), the femur (including the medulla os
sium), the musculus rectus femoris and the mammary glands.
Females At Termination of Administration Period:
Stomach: hyperplasia on anterior stomach squamous epithelium was seen in all 5 animals in the 1000
, 250 and 62.5 mg/kg group. The degree was moderate in the 1000 mg/kg group, mild in the 250 mg/
kg group and very mild or mild in the 62.5 mg/kg group. In addition, an ulcer in the anterior stomach
was seen in one animal in the 250 mg/kg group and the degree was mild. In these findings, hyperplas
ia of the anterior stomach squamous epithelium was confirmed at a significant difference compared to
the control group in the 1000, 250 and 62.5 mg/kg groups and the dose reactivity was also confirmed.
In addition, no aberrations were seen in the 1000 mg/kg group and the control group in the heart,
lungs, trachea, liver, pancreas, sublingual gland, submandibular gland, esophagus, duodenum, int
estine, ileum (including Peyer’s patch), appendix, colon, rectum, thymus, spleen, submandibular lym
ph nodes, mesenteric lymph nodes, kidneys, bladder, ovaries, uterus, pituitary gland, adrenal gland
, thyroid (including parathyroid), cerebrum, cerebellum, pons, spinal cord, sciatic nerve, eyeballs,
Harderian gland, sternum (including medulla ossium), femur (including medulla ossium), musculus r
ectus femoris (including medulla ossium) and the mammary glands.
Males after Recovery Period Terminated:
Stomach: hyperplasia of the anterior stomach squamous epithelium was seen in 6 animals in the
1000 and 250 mg/kg group; the degree was very mild or mild in the 1000 mg/kg group and extremely
mild in the 250 mg/kg group. The findings showed a significant difference compared to the control
groups in the 1000 and 250 mg/kg groups and no aberrations were noted in the stomach in the 62.5
mg/kg group even for dose reactivity.
Findings were obtained as indicated below as other changes, however, these were determined to be
one-sided incidental changes.
Testes: one-sided atrophy in the seminiferous tubule was seen in the 1000 mg/kg group, however,
during the necropsy, this was a case where a diminution of the testes and the epididymis was seen.
Epididymis: Although one-sided atrophy was seen in one case in the 1000 mg/kg group, during the n
ecropsy, this turned out to be a case where a diminution of the testes and epididymis was seen.
Females after Recovery Period Terminated:
Stomach: Hyperplasia of the anterior stomach squamous epithelium was observed in all 5 cases in
the 1000 mg/kg group. The degree was mild or very mild.
This finding was significantly different for the 1000 mg/kg group compared to the control group.
In the control group, there were no abnormalities in the stomach.
Female Parents in Mating Group:
Stomach: Hyperplasia of the anterior stomach squamous epithelium was seen in 6 cases in the 1000
mg/kg group and the degree was mild or moderate. In addition, stomach ulcers were seen in 3 cases
in the 1000 mg/kg group and the degree was extremely mild or mild. In the 1000 mg/kg group and the
control groups, no aberrations were seen in the ovaries, uterus and mammary glands.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

no hyperplasia of the anterior stomach mucus membrane was seen in the males and females in
the 1000 and 250 mg/kg groups and in the males and females in the 1000 mg/kg group when the
administration period terminated.

Reproductive function / performance (P0)

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

Number of Ruttings:
No significant difference was seen in the number of ruttings during the administration period (14 days) before mating started for the administration groups compared to the control groups.

Number of Days Required for Mating, Mating Rate, Number of Females Conceiving and Conception Rate:
The mating rate for matings within 4 days after mating started for all cases in all the groups was 100.0 % for all groups. No significant difference was seen in the number of days required for mating between the control groups for each administration group.
Only 1 female who did not conceive was seen in the control group and no significant difference was seen in the conception rate between the control groups for each administration group.

Gestation Period:
No significant difference was seen in the gestation period compared to the control groups for each administration group.
Number of Luteal Bodies, Number of Nidations and Nidation Rate:
No significant difference was seen in the number of luteal bodies, the number of nidations and the nidation rate compared to the control groups for all the administration groups.

Birth Rate, Parturition Condition and Lactation Condition:
The birth rate was 100.0 % in all of the groups.
No aberrations were seen in the parturition state and the lactation state in any of the groups.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

1

Key result

Critical effects observed:

no

System:

male reproductive system

2

Key result

Critical effects observed:

no

System:

female reproductive system

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

In each treatment group, there were no significant differences in the total number of births, number of stillborns, number of newborns on Day 0 of lactation, gender ratio on Day 0 of lactation, parturition rate, child delivery rate and birth rate compared to the control group.

Mortality / viability:

no mortality observed

Description (incidence and severity):

No significant differences were seen in the number of surviving young on day 4 of lactation, the gender ratio on day 4 of lactation and in the survival rate on day 4 of lactation in any of the groups compared to the control groups.
No epicuticle aberrations in the neonatals were seen in any of the groups.
No aberrations were seen in the general condition of the animal young in any of the groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no significant differences between males and females for each treatment group compared with the control group for mean body weight by sex, average body weight of litters and total body weight of litters between Days 0-4 of lactation.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: genital development toxicity

Target system / organ toxicity (F1)

1

Key result

Critical effects observed:

no

System:

male reproductive system

2

Key result

Critical effects observed:

no

System:

female reproductive system

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Treatment related:

no

Applicant's summary and conclusion

Conclusions:

No changes involving genital development toxicity of the parent animals which were caused by the test substance were seen in the number of ruttings, mating rate, number of days required for mating, number of females conceiving, conception rate, gestation period, delivery rate, number of luteal bodies, number of nidations, nidation rate, parturition condition and lactation condition in the administration period (14 days) before mating began.
No changes among the animal young which were caused by the test substance were seen in the total number of births of animal young, number of deaths of animal young, number neonatals on lactation day 0, the parturition rate, the young animal delivery rate, the birth rate, the gender ratio, the number of neonatals on lactation day 4, the survival rate on lactation day 4, the general condition, the body weight on lactation days 0 and 4 and the epicuticle and necropsy findings. As indicated above, the non-effective dose of the octanoic acid is thought to be less than 62.5 mg/kg/day as hyperplasia of the anterior stomach squamous epithelium was confirmed for both females and males in the 62.5 mg/kg group. A genital developmentally toxicologically non-effective dose was confirmed to be influential in all of the items for both females and males in the 1000 mg/kg group so that it is thought to be 1000 mg/kg/day.
The non-effective dose on the animal young was such that no effect was seen in any of the items in the 1000 mg/kg group so that the non-effective dose is believed to be 1000 mg/kg/day.