Carboxylic acids, C5-9

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

A study according to OECD 422 is available on the main constituent octanoic acid

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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

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.

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

Dose / conc.:

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

Dose / conc.:

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

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

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.

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: sperm measures:

not specified

Reproductive performance:

no effects observed

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.

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

Key result

Critical effects observed:

no

System:

male reproductive system

Key result

Critical effects observed:

no

System:

female reproductive system

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.

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

Key result

Critical effects observed:

no

System:

male reproductive system

Key result

Critical effects observed:

no

System:

female reproductive system

Key result

Reproductive effects observed:

no

Treatment related:

no

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.

Reference 2

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

There is one study available that assesses the possible toxic effect of the test substance after repeated oral dosing. It was performed according to GLP and internationally accepted guidelines. The experimental procedures were based on the OECD guideline 422, in which both female and male rats were treated during 5 weeks. Three doses were tested ( 62.5, 250 and 1000 mg/kg bw/day) and compared to a control group that received the vehicle only.

Concerning the parents:

No differences in body weights and food consumption were observed in treated animals compared to the control group.

No clinical signs were observed during the study.No adverse findings were recorded in clinical pathology investigations (haematology, clinical chemistry and urine analysis) apart for lymphocytosis in mid- and high dose groups. No relevant differences were recorded in the absolute and relative organ weights of treated animals. No treatment-related changes were noted at macroscopic and microscopic observations.

Concerning the pups:

Fertility index and copulatory index were unaffected by treatment. Fertility index and copulatory index were unaffected by treatment and litter data parameters and sex ratio did not show differences.

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for reproductive and developmental toxicity was considered to be the highest dose of 1000 mg/kg bw/day.

No test reports are available that address the repeated dose toxicity of the test substance when exposure occurs via the inhalation or the dermal pathway. However, based on the outcome of the available study data, it is not deemed necessary to evaluate the other routes in more detail.

Short description of key information:
A NOAEL of 1000 mg/kg bw/d was determined for the test item based on effects observed in a combined repeated dose / reprotox screening test (OECD422).

Justification for selection of Effect on fertility via oral route:
Well documented study according to GLP and internationally accepted guidelines.

Effects on developmental toxicity

Description of key information

There is one study available describing developmental toxicity effects (Azuka and Daston, 2014) on the read-across substance Trimethylolpropane Caprylate Caprate (CAS11138-60-6). Based on the results of that study there is no indication of any potency to cause developmental effects as the determined NOAEL for developmental effects was > 2000 mg/kg bw.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Hypothesis for the analogue approach: This read-across is based on the hypothesis that source and target substances have similar toxicological properties because, following oral intake, the source substances hydrolyse in the gut to non-common products predicted to have no toxicological effect (metabolic approach). The target substance is n-nonanoic acid, a saturated linear, medium-chain length carboxylic acid. The prediction is limited for saturated linear, medium-chain length carboxylic acids as source substances and for systemic toxicity endpoints, e.g. repeated dose toxicity and toxicity to reproduction. The prediction is supported by valid toxicological data on the substances and their hydrolysis products, based on known rapid and extensive hydrolysis and subsequent metabolism. For read across justification, see the atteched file in endpoint 13 ( Analogue approach justification): Acceptable, well-documented publication which meets basic scientific principles

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

2 doses tested

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HRP Inc, Denver, PA, USA
- Age at study initiation: 5.5 to 6.5 months
- Weight at study initiation: 330 to 4446 g on GD 0
- Housing: singly
- Diet: ad libitum except during dose administration
- Water: ad libitum except during dose administration
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
air-conditioned rooms

Route of administration:

other: intravenous infusion (ear vein)

Vehicle:

soya oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): for reasons of solubility
- Concentration in vehicle: 3.1 ratio of MCT:LCT from soya oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability was analytically determined which implies analytical verification of composition and doses. Methods were not described.

Details on mating procedure:

Time-mated animals; no further details described.

Duration of treatment / exposure:

GD 7 through 19

Frequency of treatment:

daily, 5h/day

Duration of test:

until GD 29

Remarks:

Doses / Concentrations:
0, 1, and 4.28 g/kg bw and day
Basis:
other: i.v. dose

No. of animals per sex per dose:

15

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: 4.28% was the highest dose in preclinical studies that did not produce narcosis

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

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

BODY WEIGHT: Yes
- Time schedule for examinations: pre-dose and on GD5 through GD20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n. a.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n.a.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes

Statistics:

The litter was the experimental unit for evaluation and all comparisons were made with the control group. Amongst other calculations, ANOVA followed by Dunnett's test was used to analyse body weights, feed consumption, caesarian section data. Foetal abnormality data were analysed using the Cochran.-Armitage test and the Fisher-Irwin exact test.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
All low-dose animals survived whereas one control and one high-dose rabbit were sacrificed after aborting on GD 20; the treated animal had the lowest feed consumption in the group. The abortion was inside the historical control range and there were no remarkable necropsy findings for either animal.
The only clinical sign noted was that 3/15 animals had no fecal output for 1 day during the treatment period. Body weights were comparable, but feed consumption and body weight gain were significantly reduced (p<0.01) during the treatment period (GD 12 through GD20) and also in the recovery period thereafter (until GD 29). Reduced feed consumption was expected due to the high caloric nature of the test material.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: nonanoic acid

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At the low dose (1000 mg/kg bw and day) foetal postimplantation was slightly increased and the mean body weight was slightly reduced without gaining a level of significance This was, however, the case at the high dose where early and late resorptions were clearly increased; as a consequence, the number of live foetuses was decreased (p<0.01), and the mean foetal weight was significantly decreased (p<0.01); cf. table 3. The incidence of external, soft tissue and skeletal malformations was increased at the high dose (statistically significant); tables 3 and 4.
The incidence of soft tissue findings was also increased in low dose foetuses, but without gaining a level of significance (tables 3 and 4) when individual malformations were analysed statistically. Similarly, skeletal findings were increased in the low dose group without gaining a level of significance (table 4).

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: nonanoic acid

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

 

 

 

Conclusions:

Intravenous doses of MCT (up to 4280 mg/kg bw and day, GD 5-20) caused maternal effects at the high dose, but no developmental toxicity or teratogenicity. The NOAEL was therefore 1000 mg/kg bw and day for maternal toxicity and 4280 mg/kg bw and day for developmental toxicity and teratogenicity.

Executive summary:

Time-mated rabbits (15/group) received intravenous infusions into the ear vein (duration 5 h/day) during Gestation Days 7 through 19 of a 20% lipid emulsion that contained a ratio of 3:1 of MCT and Long Chain Triglycerides (LCT) from soy bean oil at doses of 1 and 4.28 g/kg bw and day. Controls received 0.9% saline. Medium chain fatty acids are 6 to 12 carbons in length. The 20% lipid emulsion is composed primarily of 8- and 10-carbon fatty acids, with only traces of 6- and 12-carbon fatty acids. The does were observed for clinical signs and sacrificed at termination, the does and of the uterus content were examined.

No treatment-related mortalities were noted. The only clinical sign was a reduced faecal output of 3/15 does on one day. Body weight change and feed intake was significantly reduced (p<0.01) in high-dose rabbits which was expected because of the high caloric test material. No adverse effect was noted in does at 1000 mg/kg bw and day, hence this was considered to represent the maternal NOAEL value.

 

Foetal findings: at the low dose (1000 mg/kg bw and day) foetal postimplantation loss was slightly increased and the mean foetal weight was slightly reduced without gaining a level of significance. This was, however, the case at the high dose where early and late resorptions were clearly increased; consequently, the number of live foetuses was decreased (p<0.01), and the mean foetal weight was significantly decreased (p<0.01). The incidence of external (p<0.05), soft tissue and skeletal malformations (p<0.05) was increased at the high dose.

The incidence of soft tissue findings was also increased in low dose foetuses, but without gaining a level of significance when individual malformations were analysed statistically. Similarly, an increase of skeletal findings was seen in the low dose group below a level of significance. Based on these findings the NOAEL for developmental toxicity was set at 1000 mg/kg bw and day, and the authors assume that the findings at 4280 mg/kg bw and day are attributable to the low feed consumption and malnutrition rather than a direct teratogenic effect of the test material (Henwood et al. 1997).

The study is considered to be valid. The intravenous route was used because the parenteral route is used in patients that cannot meet their nutritional needs by the conventional oral route, and 1000 mg/kg bw and day is the approximate clinical dose, whereas 4280 mg/kg bw and day is reportedly the highest preclinical dose that did not cause narcosis.

The study can be used for the assessment of n-nonanoic acid in a way as this has been described in the repeated dose toxicity section for the studies of Matulka and Webb who also administered triglycerides as test material. In brief,

-         the metabolism of straight chain fatty acids of 8 to 10 carbons including nonanoic acid is very similar and includes rapid degradation via ß-oxidation, which justifies cross reading form C8 and C10 fatty acids to C9 nonanoic acid.

-         The fatty acid composition of the test material of this study (Henwood et al., 1997) was almost exclusively C8 and C10, with traces of C6 and C12.

-         The mean molecular weight of the triglycerides is calculated assuming that traces are negligible, and that C8 and C10 are present in equimolar proportions (this was not specified in the publication). Then, the mean triglyceride molecular weight is 596, and the two fatty acids represent 42.5% each of the molecular weight, and also of the dose, i.e. approx. 425 mg of C8 and also of C10 fatty acid/kg bw and dose.

In a weight of evidence approach, this result can be used to assess n-nonanoic acid: NOAEL approx. 425 mg/kg bw and day.

 

 

 

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Hypothesis for the analogue approach: This read-across is based on the hypothesis that source and target substances have similar toxicological properties because, following oral intake, the source substances hydrolyse in the gut to non-common products predicted to have no toxicological effect (metabolic approach). The target substance is n-nonanoic acid, a saturated linear, medium-chain length carboxylic acid. The prediction is limited for saturated linear, medium-chain length carboxylic acids as source substances and for systemic toxicity endpoints, e.g. repeated dose toxicity and toxicity to reproduction. The prediction is supported by valid toxicological data on the substances and their hydrolysis products, based on known rapid and extensive hydrolysis and subsequent metabolism. For read across justification, see the atteched file in endpoint 13 ( Analogue approach justification): Acceptable, well-documented publication which meets basic scientific principles

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

2 doses tested

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

other: Crl:CD BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Portage, MI, USA
- Age at study initiation: no data
- Weight at study initiation: 150 to 200g
- Housing: singly
- Diet: ad libitum except during dose administration
- Water: ad libitum except during dose administration
- Acclimation period: 2

ENVIRONMENTAL CONDITIONS
air-conditioned rooms

Route of administration:

other: intravenous infusion (caudal vein)

Vehicle:

soya oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): for reasons of solubility
- Concentration in vehicle: 3.1 ratio of MCT:LCT from soya oil

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability was analytically determined which implies analytical verification of composition and doses. Methods were not described.

Details on mating procedure:

Time-mated animals; no further details described.

Duration of treatment / exposure:

GD 6 through 15

Frequency of treatment:

daily

Duration of test:

until GD 20

Remarks:

Doses / Concentrations:
0, 1, and 4.28 g/kg bw and day
Basis:
other: i.v. dose

No. of animals per sex per dose:

25

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: 4.28% was the highest dose in preclinical studies that did not produce narcosis

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

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

BODY WEIGHT: Yes
- Time schedule for examinations: pre-dose and on GD5 through GD20

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): n. a.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): n.a.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

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

Statistics:

The litter was the experimental unit for evaluation and all comparisons were made with the control group. Amongst other calculations, ANOVA followed by Dunnett's test was used to analyse body weights, feed consumption, caesarian section data. Foetal abnormality data were analysed using the Cochran.-Armitage test and the Fisher-Irwin exact test.

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Tail lesions such as discoloration and ulceration were seen in treated groups (incidences 1/25, 14/25, and 23/29 in the control low- and high-dose groups, respectively. Findings ranged from mild to severe with some necrosis and partial tail loss. This was related to extravasation of the lipid test article.
Body weight was comparable between controls and low-dose animals; reduced body weight and feed consumption was noted in high-dose rats. After cessation of treatment, feed consumption increased. Necropsy revealed tail effects in the low dose group and especially in the high dose animals. . Further, there was a trend in the high-dose group, towards enlarged lymph nodes, enlarged spleen, hydronephrosis/enlarged renal pelvis, small thymus, and small red lung foci. See table below for further details.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

ca. 4 280 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Dose descriptor:

NOAEL

Effect level:

1 820 mg/kg bw/day (actual dose received)

Based on:

other: estimated NOAEL for octa- and decanoic acid. Read acorss: also of nonanoic acid

Basis for effect level:

other: developmental toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There were no significant group differences in preimplantation or postimplantation loss or in the mean percentage of live or resorbed foetuses; no dead foetuses were present. Mean foetal sex ratios of the test article-treated groups were comparable with those of controls. There were no apparent effects on mean foetal body weight (combined, males, or females). Finally, there were no test article-related foetal external, soft tissue, or skeletal observations.

Dose descriptor:

NOAEL

Effect level:

ca. 4 280 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

ca. 1 820 mg/kg bw/day (actual dose received)

Based on:

other: estimated NOAEL for octa- and decanoic acid. Read acorss: also of nonanoic acid

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

 

 

 

Conclusions:

Intravenous doses of MCT (up to 4280 mg/kg bw and day, GD 5-20) caused maternal effects at the high dose, but no developmental toxicity or teratogenicity. The NOAEL was therefore 1000 mg/kg bw and day for maternal toxicity and 4280 mg/kg bw and day for developmental toxicity and teratogenicity.

Executive summary:

Time-mated rats (25/group) received intravenous infusions into the caudal vein (duration 4h/day) during gestation Days 6 through 15 of a 20% lipid emulsion that contained a ratio of 3:1 of MCT and Long Chain Triglycerids (LCT) from soy bean oil at doses of 1 and 4.28 g/kg bw and day. Controls received 0.9% saline). Medium chain fatty acids are 6 to 12 carbons in length. The 20% lipid emulsion is composed primarily of 8- and 10-carbon fatty acids, with traces of 6- and 12-carbon fatty acids. The dams were observed for clinical signs and sacrificed at termination. Examinations of the dams and of the uterus content were performed.

No clinical signs or mortalities were noted. Changes in high-dose rats (enlarged lymph nodes, spleen, and renal pelvis; small thymus, small red lung foci) were considered to be related to treatment, hence the NOAEL was 1 000 mg/kg bw and day for maternal toxicity. As to the foetuses, there were no significant differences compared to controls regarding early or late resorptions, post implantation loss, dead or alive foetuses, mean uterine weight and foetal weight (though there was a trend towards lower weights at the high dose), and there were no external, soft tissue or skeletal changes that could be related to treatment. Hence, the NOAEL for developmental toxicity and teratogenicity was 4280 mg/kg bw and day (Henwood et al., 1997).

The study is considered to be valid. The intravenous route was used because the parenteral route is used in patients that cannot meet their nutritional needs by the conventional oral route, and 1000 mg/kg bw and day is the approximate clinical dose, whereas 4280 mg/kg bw and day is reportedly the highest preclinical dose that did not cause narcosis.

The study can be used for the assessment of n-nonanoic acid in a way as this has been described in the repeated dose toxicity section for the studies of Matulka and Webb who also administered triglycerides as test material. In brief,

-         the metabolism of straight chain fatty acids of 8 to 10 carbons including nonanoic acid is very similar and includes rapid degradation via ß-oxidation, which justifies cross reading form C8 and C10-fatty acids to C9 nonanoic acid.

-         The fatty acid composition of the test material of this study (Henwood et al., 1997) was almost exclusively C8 and C10, with traces of C6 and C12.

-         The mean molecular weight of the triglycerides is calculated assuming that traces are negligible, and that C8 and C10 are present in equimolar proportions (this was not specified in the publication). Then, the mean triglyceride molecular weight is 596, and the two fatty acids represent 42.5% each of the molecular weight, and also the dose, i.e. approx. 1820 mg of C8 and also of C10 fatty acid/kg bw and dose.

In a weight of evidence approach, this result can be used to assess n-nonanoic acid: NOAEL approx.. 1820 mg/kg bw and day.

 

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1998

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

not specified

Limit test:

no

Specific details on test material used for the study:

Cekanoic C8 acid

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Kingston Facility, Stone Ridge, NY, USA
- Age at study initiation: no data
- Weight at study initiation: 150 to 200g
- Housing: singly
- Diet: ad libitum except during dose administration
- Water: ad libitum except during dose administration
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
Room temperature: 68-76 °F and Relative humidity: 40-70% were monitored daily
Suspended stainless-steel cages maintained on a 12-hour photoperiod.

Route of administration:

oral: gavage

Vehicle:

corn oil

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

A range-finding study was conducted by esposing confirmed mated females (7 per dose group) to 0, 50, 200, 400, 800 and 1000 mg/kg/day of isooctanoic acid from GD6 to GD15. Maternal toxicity was demonstrated at the two highest dosages by clinical signs in the majority of the treated animals (rales, stool abnormlities, abdominal/anogenital staining), reduced food consumption and weight loss during the treatment period. There were no effects on embryo/fetal viability or evident malformations, only a weigh reduction of anout 10%.
Based on these findings, the doses selected for the definitive study were 200, 400 and 800 mg/kg/day.

Details on mating procedure:

Individual females after acclimation period were housed overnight with a single untreated male of the same stock. The observation of a copulatory plug and/or sperm in a vaginal rinse was considered evidence of successful mating.

Duration of treatment / exposure:

Mated females were dosed by oral gavage with the test material formulated in corn oil once daily from GD6 trough GD 15.

Frequency of treatment:

Once a day from GD6 to GD15

Duration of test:

21 days

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Maternal examinations:

Maternal body weight and food consumption were measured on GD 0, 6, 9, 12, 15, 18 and 21.

Ovaries and uterine content:

On GD 21, all females were euthanized by inhalation of CO2 and the reproductive tract of each dam was exposed by an abdominal incision. The uteri with ovaries were removed and weighed, and the number of corpora lutea in each ovary was counted. The uterus was opened, and the number and location of implantation sites, resorptions, and live and dead fetuses were recorded. Non gravid uteri were stained with ammonium sulfide to confirm pregnancy status.

Fetal examinations:

All live fetuses were weighed, sexed externally and examined externally for gross malformations. The viscera of approximately one-half of the live fetuses from each litter were immediately examined for abnormalities. These fetuses were then decapitated and their heads were fixed in Bouin's solution for subsequent examination. The remaining fetuses were eviscerated and processed for skeletal staining with Alizarin red-S stain and examined for the presence of malformations and ossification variations.

Statistics:

Maternal Data
Maternal body weight data and body weight change, food consumption and uterine data (i.e., corpora lutea, implants, resorptions) were analyzed using Bartlett's Test of homogeneity of Variance. If the variances were equal, the groups were compared using a standard one-way analysis of variance (ANOVA). If significant differences among the means were indicated, Dunnett's Test was performed to determine which treated groups differed from control. In addition to the ANOVA, a standard regression analysis for linear response in the dose group was performed. If the group did not have equivalent variances a Kruskal-Wallis test was used to assess differences in the group means. If the means were were different,k Dunn's Summed Rank Test was used to determine which treatment groups differed significantly from contro. Also Jonckheere's Test for monotonic trend in the dose-response was performed. Bartett's Test for equal variance was conducted at the 1 % level of significance. All other tests were conducted at the 5% and 1% levels of significance.

Fetal Data
Fetal weight was analyzed by a nested analysis of covariance with fetuses nested within dams and with dams nested within doses and litter size as the covariant. If differences in groups were identified, the Least Significant Difference (LSD) technique was used to determine which groups differed from the control group. Foetal abnormality data were analysed using the Cochran.-Armitage test and the Fisher-Irwin exact test. All tests were reported at the 5% or 1% level of significance

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

All females survived to scheduled study termination on GD21. Clinical signs were limited to anogenital staining and alopecia in 6 of 25 dams in the 800 mg/kg/day-dose group following dose initiation. There were no postmortem findings judged as treatment-related.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no significant differences in absolute maternal body weight in any of the treatment group at any time during the study. There were however significant reductions in maternal body weight gain in the high-dose (800 mg/kg/day) group compared with controls during GD 6-9 and throughout the entire period (GD 6-15). No differences at lower dosages.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean food consumption, during GD 6-9 and the overall treatment period (GD 6-15) was significantly reduced at 800 mg/kg/day compared with the controls. However, for the overall gestation period (GD 0-21, mean food consumption in all treatment groups was essentially equivalent. No differences at lower dosages.

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:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

At 800 mg/kg/day maternal body weight, food consumption and clinical observations were recorded throughout gestetian. No effect obtained with lower dosages.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Details on maternal toxic effects:

There were no significant differences between any of the treatment groups in the total number of corpora lutea, implantation sites, live fetuses, resorptions or dead fetuses per dam (see table 1). Accordingly, there were no differences in the percentage of pre- or post-implantation losses. There were no statistically significant differences in fetal body weights in any weights appeared to trend slightly downward, none of the female body-weight means fell outside the laboratory's historical control range. There were also no significant differences in the sex ratio of fetuses in any treatment group.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

There was a significant increase in the incidence of skeletal variations in the 800 mg/kg/day group compared with the control. The incidence (4.4%) was within the historical control range of variations/litter for this laboratory (0-6.3%). One fetus each from the 200 mg/kg and 800 mg/kg dose groups had malformed sternebrae.There were linear trends observed in the incidence of hypoplastic skull bones and rudimentary lumbar ribs on a per fetus basis only. However, the only significant difference found in pairwise comparisons of means was in rudimentary lumbar ribs (

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Visceral malformations observed consisted of folded retina, dilated cerebral ventricles and anopthalmia in one fetus each from the control, 200 and 800 mg/kg grouos, respectively (see Table 3).

Other effects:

no effects observed

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: absence of biologically significant fetal effects

Abnormalities:

no effects observed

Developmental effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

yes

Reproductive parameters in rats exposed to isooctanoic acid on gestation days 6 -15

Table 1*	Dose (mg/kg/day)
	0	200	400	800
N° of cesarean sections **	22	23	23	25
Corpora lutea/dam	17.9 +/- 3.3	17.3 +/- 2.5	17.2 +/- 1.7	17.9 +/- 2.2
Implantations/litter	16.4 +/- 2.0	16.5 +/- 2.2	16.4 +/- 2.4	16.3 +/- 2.4
Pre-implantation loss (%)***	7.0 +/- 11.0	3.7 +/- 5.2	4.8 +/- 10.7	8.6 +/- 11.2
Viable fetuses/litter	15.6 +/- 1.7	15.7 +/- 2.5	15.2 +/- 2.5	15.4 +/- 2.4
Dead fetuses/litter	0	0	0	0
Resorptions/litter	0.8 +/- 0.9	0.8 +/- 1.1	1.2 +/- 1.4	0.9 +/- 0.9
Post-implantation loss (%)****	4.4 +/- 4.9	4.8 +/- 7.3	7.3 +/- 8.5	5.4 +/- 5.4
Fetal body weight (g)
Males	5.49 +/- 0.46	5.55 +/- 0.35	5.53 +/- 0.37	5.41 +/- 0.33
Females	5.28 +/- 0.32	5.20 +/- 0.37	5.18 +/- 0.38	5.09 +/- 0.41
Fetal sex distribution
Males	8.00 +/- 1.61	7.91 +/- 2.04	8.00 +/- 2.71	7.24 +/- 1.85
Females	7.62 +/- 2.16	7.83 +/- 2.12	7.17 +/- 2.46	8.20 +/- 2.02

*Pregnant dams were esposed daily by gavage o the indicated dse and assessments were performed on gestation day 21. Values are given as mean +/- SD;

**One low-dose and 3 control dams were not pregnant; one low-dose and two mid-dose dams delivered early and were excluded;

***(Corpora lutea – implantation sites)/corpora lutea x 100;

****(Implantation sites – live fetuses)/Implantation sites x 100.

Summary of fetal findings in rat exposed to isooctanoic acid on gestation days 6-15

Table 2*	Dose (mg/kg/day)
	0	200	400	800
N° of litters examined	22	23	23	25
N° of fetuses examined externally	328	326	349	370
N° of fetuses examined viscerally	169	179	175	196
N° of fetuses examined skeletally	163	172	174	190
External observations (stunted
Stunted (< 4 g), % fetuses (litters)	0.6 (9.5)	0.3 (4.3)	0.3 (4.3)	1.0 (12.0)
Malformations
External, % fetuses (litters)	0 (0)	0 (0)	0 (0)	0 (0)
Visceral, % fetuses (litters)	0.6 (4.5)	0.6 (4.3)	0 (0)	0.5 (4.0)
Skeletal, % fetuses (litters)	0 (0)	0.6 (4.5)	0 (0)	0.5 (4.0)
Total malformations/litter^a	0.05 +/- 0.22	0.09 +/- 0.29	0.00 +/- 0.00	0.08 +/- 0.28
Total affected/litter^b	0.9 +/- 0.9	0.9 +/- 1.2	1.2 +/- 1.4	1.0 +/- 0.9
Variations
External, % fetuses (litters)	0 (0)	0 (0)	0 (0)	0 (0)
Visceral, % fetuses (litters)	0 (0)	0 (0)	0 (0)	0 (0)
Skeletal, % fetuses^c(litters)	33.7 (90.5)	33.1 (81.8)	42.5 (95.7)	58.4**(88.0)
Total variations/litter^cd	2.6 +/- 1.9	2.6 +/- 2.1	3.2 +/- 2.3	4.4 +/- 2.6***

*Pregnant dams were exposed daily by gavage to the indicated dose and assessments were performed on gestation day 21. Percentage data were calculated as: N° affected/N° examined x 100;

^aN° of fetuses/litter with one or more external, visceral or skeletal malformations (mean +/- SD);

^bTotal number of resorptions, dead and malformed fetuses per litter (mean +/- SD);

^cLinear trend in dose groups by Armitage’s test;

^dN° of fetuses/litter with one or more external, visceral or skeletal variations (mean +/- SD);

**Significantly different from control group; p<0.05;

*** Significantly different from control group; p<0.01.

Specific malformations and variations observed in fetuses exposed to isooctanoic acid on gestation days 6-15

Table 3*	Dose (mg/kg/day)
	0	200	400	800
Visceral malformations
Anopthalmia	0 (0)	0 (0)	0 (0)	0.5 (4.0)
Folded retina	0.6 (4.5)	0 (0)	0 (0)	0 (0)
Dilated cerebral ventricles	0 (0)	0.6 (4.3)	0 (0)	0 (0)
Skeletal malformations
Malformed sternebrae	0 (0)	0.5 (4.3)	0 (0)	0.5 (4.0)
Skeletal variations
Skull bones hypoplastic^a	4.3 (27.3)	2.9 (21.7)	8.0 (21.7)	8.4 (28.0)
Hyoid unossified	0 (0)	0 (0)	0 (0)	0.5 (4.0)
Ossification site between parietals and interparietal	0 (0)	0.6 (4.3)	0 (0)	0.5 (4.0)
Supraoccipital misshapen	0 (0)	0 (0)	0 (0)	1.6 (4.0)
Sternebrae
Hypoplastic	0.6 (4.5)	1.2 (4.3)	0 (0)	0 (0)
Unossified	0.6 (4.5)	0.6 (4.3)	3.4 (13.0)	0.5 (4.0)
Misshapen	0.6 (4.5)	0 (0)	0 (0)	0 (0)
Bifid	0.6 (4.5)	0 (0)	0 (0)	0 (0)
Ribs
Rudimentary lumbar^a	28.8 (77.3)	29.1 (78.3)	31.6 (82.6)	48.4***(88.0)
Rudimentary cervical	0 (0)	0 (0)	3.4** (21.7)**	0.5 (4.0)
Well-formed lumbar	0.6 (4.5)	1.2 (8.7)	0 (0)	3.7 (20.0)
Bent thoracic	0 (0)	0 (0)	1.1 (4.3)	0 (0)
Vertebrae
Supernumerary presacral	1.2 (9.0)	0 (0)	0 (0)	4.2 (12.0)
Bifid centra	0.6 (4.5)	0.6 (4.3)	0 (0)	0 (0)

*Pregnant dams were exposed daily by gavage to the indicated dose and assessments were performed on gestation day 21. Data represent the percentage of fetuses (litters) displaying the anomaly per dose group. Percentage data were calculated as: N° affected/N°examined x 100. Number of fetuses and litters examined are given in Table 2;

^aLinear trend in dose groups are obtained by Armitage’s test;

**Significantly different from control group; p<0.05;

*** Significantly different from control group; p<0.01.

Conclusions:

Isooctanoic acid (CAS RN 25103-52-0) is a complex isomeric substance containing several aliphatic carboxylic acids, primarily dimethyl hexanoic acid. Because Cekanoic C8 acid is a structural isomer of octanoic acid, its potential for developmental toxicity was investigated in CD (Sprague-Dawley) rats. It was administered by oral gavage to 25 confirmed-mated females at doses of 0, 200, 400, and 800 mg/kg/day on gestation days (GD) 6-15, based on a range-finding experiment. Maternal body weights, food consumption, and clinical observations were recorded throughout gestation. On GD 21, cesarean sections were performed and the uterine contents removed and subjected to conventional teratological evaluation. At 800 mg/kg/day, maternal body weight gain and food consumption were reduced during the exposure period, and clinical signs were evident. There were no significant differences in fetal weight, malformation incidence, or fetal viability in any of the experimental groups. There was a statistically significant increase in the incidence of total variations in the 800-mg/kg/day group, which was within the historical control range of the laboratory and not considered biologically significant. These results indicate that, isooctanoic acid was not teratogenic or a selective developmental toxicant in rats. This study established a maternal no-observable-adverse-effect level (NOAEL) at 400 mg/kg/day and a developmental NOAEL at 800 mg/kg/day.

Reference 4

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions; few details on test substance given, no analysis of the test compound

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

yes

Remarks:

few details on test substance given, no analysis of the test compound

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Kingston, NY
- Age at study initiation: young adult
- Weight at study initiation: Mean of the maternal body weight: 226 g (Vehicle), 225 g (200 mg/kg bw/day), 227 g (600 mg/kg bw/day), 226 g (2000 mg/kg bw/day)
- Fasting period before study: No
- Housing: Virgin females were cohabitated with singly-housed male rats, one male per female rat for a maximum of 5 days and returned to individual housing in stainless steel wire-bottomed cages after mating.
- Diet: Certified Rodent Diet No. 5002 (PMI Feeds Inc. St.Louis, MO), ad libitum
- Water: water passaged through a reverse osmosis membrane with chlorine added as a bacteriostat, ad libitum
- Acclimation period: yes, period not mentioned

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

dermal

Vehicle:

corn oil

Details on exposure:

- Doses: 0, 200, 600, and 2,000 mg/kg/day.

- Dose formulation: 0 (vehicle only), 100, 300, and 1,000mg/mL

- Dosage volume: 2 mL/kg.

- Application of dose: The dosage amount was applied directly to the clipped area on the dorsum of the rat at approximately the same time each day and spread uniformly over the area with a glass rod. The skin application site was occluded during treatment with a gauze pad secured with Vetrapt or Micropores tape to prevent oral ingestion and to minimize loss of material from under the patch.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Treatment on Gestation Days (GD) 6 - 15

Frequency of treatment:

daily

Duration of test:

Termination of the study by CO2 inhalation on GD 20.

No. of animals per sex per dose:

25 females per group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose dependent occurrence of skin irritation. Higher levels than 2000 mg/kg bw/day might be expected to produce marked irritation thereby compromising the interpretaion of developmental results.
- Rationale for animal assignment (if not random): Computer-generated randomization by weight (Barlett´s test for homogeneity) such that the groups were not statistically different (5% significance level) from each other.

Maternal examinations:

CAGE SIDE OBSERVATIONS
- Time schedule: Animals were checked for mortality twice daily during the treatment period and daily thereafter.


DETAILED CLINICAL OBSERVATIONS
- Time schedule: Animals were checked for signs of reaction to treatment and/or symptoms of illness once daily before treatment, approx. 60 min after treatment during the dosing period. The dosing site was examined daily prior to substance application for signs of skin irritation according to Draize.


BODY WEIGHT
- Time schedule for examinations: Recorded on GD 0 and daily during the treatment period.


FOOD CONSUMPTION AND COMPOUND INTAKE
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg b.w./day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


POST-MORTEM EXAMINATIONS
- Sacrifice on gestation day # 20
- Organs examined: The uterus, uterine contents, position of the fetuses in the uterus and number of corpora lutea. Number and distribution of intrauterine implantations were classified as live or death fetuses, late intrauterine deaths (resorptions) and early intrauterine resorption sites. Live fetuses were sexed and further examined (see below).

Ovaries and uterine content:

- Gravid uterus weight
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions

Fetal examinations:

- External examinations: all per litter
- Soft tissue examinations: half per litter
- Skeletal examinations: half per litter
- Head examinations: half per litter (the heads of the animals used for soft tissue examinations)

Statistics:

Clinical observation and other proportion data were analyzed using the Variance Test for Homogeneity of the Binomial Distribution (Snedecor and Cochran, 1967). Quantitative continuous data (e.g., maternal body weights, body weight changes, feed consumption values, litter averages for percent male fetuses, percent resorbed conceptuses, fetal body weights, fetal anomaly data, and fetal ossification data) were analyzed using Bartlett’s Test
for Homogeneity of Variance (Sokal and Rohlf, 1969) and the Analysis of Variance (Snedecor and Cochran, 1967) when Bartlett’s Test was not significant (p40.05). If the Analysis of Variance was significant (pr0.05), Dunnett’s Test (Dunnett, 1955) was used to identify the statistical significance of the individual groups. If the Analysis of Variance was not appropriate, i.e., Bartlett’s Test was significant (pr0.05), the Kruskal-Wallis Test (Sokal and Rohlf, 1969) was used when < 75% ties were present. In cases where the Kruskal-Wallis Test was statistically significant (p<0.05), Dunn’s Method of Multiple Comparisons (Dunn, 1964) was used to identify the statistical significance of the individual groups. If there were 475% tied, Fisher’s Exact Test (Siegel, 1956) was used to analyze the data. Count data obtained at Caesarian-sectioning of the dams were evaluated using the procedures described above for the Kruskal-Wallis Test.

Clinical signs:

no effects observed

Details on maternal toxic effects:

Maternal toxic effects:yes. Remark: local irritation

Details on maternal toxic effects:
The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain or feed consumption. Two animals in the control group and one animal in the high-dose group died within 6 h after first application; these were not considered to be treatment related and the animals were replaced. One dam of the mid-dose goup (1/25) having a litter consisting of seven early resorptions was pointed out as single non-dosage dependent event and to be within the ranges observed historically at the test facility.
Necropsy findings were limited to skin flaking and scabbing first identified in life and observations related to wearing the Elizabethan collar (local alopecia, chromorhinorrhea, and neck lesions).

Key result

Dose descriptor:

NOAEL

Effect level:

> 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: developmental toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Dose descriptor:

NOAEL

Remarks:

local

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Fetal body weight changes:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:

There were no significant differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. The observed effects in fetuses were dose-independent and regarded to be sporadic.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day

Based on:

test mat.

Sex:

not specified

Basis for effect level:

fetal/pup body weight changes

changes in postnatal survival

external malformations

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Skin reaction observations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Maximum possible incidences^a	375/25	375/25	375/25	375/25
Erythema
Total	0/0	2/1	22/4	91/13^b
Grade 1	0/0	2/1	10/4	81/13^b
Grade 2	0/0	0/0	4/1	10/4^b
Flaking
Total	11/3	15/2	55/6	170/17^b
Grade 1	11/3	9/2	27/5	61/14^b
Grade 2	0/0	6/1	19/4	71/14^b
Grade 3	0/0	0/0	9/1	38/7 ^b
Edema
Total	0/0	0/0	23/4	83/11^b
Grade 1	0/0	0/0	18/4	59/11^b
Grade 2	0/0	0/0	5/1	24/6^b
Scab	0/0	0/0	6/2	19/4

a:       Maximum incidence : Days x rats from first treatment on GD 6 through sacrifice on GD 20 divided by the number of rats examined per group on GD 6-20

b:        Significantly different from vehicle control group value (p≤0.01)

 

Table 2: Maternal reproductive, litter, and fetal alteration observations: Caesarian-Section results on GD 20

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Rats pregnant and sectioned on Day 20 of gestation (n)	25	23	22^b	24
Corpora lutea/dam	16.4	16.6	16.9	16.5
Implantation sites/litter	15.0	15.4	14.9	14.2
Litter size
Live fetuses/litter	14.6	14.6	14.0	13.3
Live fetuses (n)	364	335	308	320
Dead fetuses (n)	0	0	0	0
Resorptions	0.4	0.9	0.9	0.9
Early (n)	10	20	19	21
Late (n)	1	0	0	0
Dams with any resorptions n(%)	9 (36)	11 (48)	15 (68)	11 (46)
% resorbed/litter	2.9	5.4	5.8	5.0
% male/litter	51.3	50.8	48.1	47.7
Live fetal body weight (g/litter)	3.68	3.62	3.69	3.75
Male	3.77	3.68	3.82	3.85
Female	3.58	3.56	3.58	3.65
Fetuses evaluated (n)	364	335	308	320
Litters with any alterations observed n(%)	10 (40)	8 (35)	14 (64)	7 (25)
Fetuses with any alterations observed n(%)	13 (3.5)	10 (3.0)	20 (6.5)	9 (2.0)
% fetuses/litter with any alterations observed	3.5	2.9	6.8^c	2.7

b:       Excludes values for one dam, which had a litter consisting of seven early resorptions.

c:       Significantly different from vehicle control group value (p≤0.05)

Table 3: Fetal evaluations

	0 mg/kg bw/d	200 mg/kg bw/d	600 mg/kg bw/d	2000 mg/kg bw/d
Litters evaluated	25	23	22^b	24
Fetuses evaluated	364	335	308	320
Live	364	335	308	320
Fetal gross external alterations	364	335	308	320
Tail: kinked
Litter incidence, n (%)	0(0)	1 (4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.3)	0(0)	0(0)
Body: hematoma
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	0(0)
Fetal incidence, n (%)	1 (0.3)	0(0)	0(0)	0(0)
Fetal soft tissue alterations, evaluations	174	162	149	155
Vessels: umbilical artery descended to the left of urinary bladder
Litter incidence, n (%)	2(8.0)	3(13.0)	2(9.1)	2(8.3)
Fetal incidence, n (%)	2(1.1)	3(1.8)	3(2.0)	2(1.3)
Vessels: apparent additional umbilical artery descended left of the bladder
Litter incidence, n (%)	0(0)	0(0)	1(4.5)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	1(0.7)	0(0)
Fetal skeletal alterations, evaluations	190	173	159	165
Cervical vertebrae: cervical rib present at 7th cervical vertebrae
Litter incidence, n (%)	2(8.0)	1(4.3)	1(4.8)	0(0)
Fetal incidence, n (%)	2(1.0)	2(1.2)	1(1.2)	0(0)
Thoracic vertebrae: centrum, bifid
Litter incidence, n (%)	1(4.0)	1(4.3)	5(22.7)	0(0)
Fetal incidence, n (%)	1(0.5)	1(0.6)	5(3.1)^a	0(0)
Lumbar vertebrae: centrum, bifid
Litter incidence, n (%)	0(0)	1(4.3)	0(0)	0(0)
Fetal incidence, n (%)	0(0)	1(0.6)	0(0)	0(0)
Ribs: wavy
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	1(4.2)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	1(0.5
Sternal centra: 1st, not ossified
Litter incidence, n (%)	1(4.0)	0(0)	0(0)	2(8.3)
Fetal incidence, n (%)	1(0.5)	0(0)	0(0)	2(1.3)
Sternal centra: 1st, incompletely ossified
Litter incidence, n (%)	3(12.0)	3(13.0)	2(5.1)	1(4.2)
Fetal incidence, n (%)	4(2.1)	4(2.3)	2(1.2)	1(0.6)
Pelvis: pubis, incompletely ossified
Litter incidence, n (%)	3(12.0)	0(0)	4(18.2)	3(12.5)
Fetal incidence, n (%)	3(1.6)	0(0)	5(3.1)	3(1.8)
Pelvis: ischium, incompletely ossified
Litter incidence, n (%)	0(0)	0(0)	2(9.1)	0(0)
Fetal incidence, n (%)	0(0)	0(0)	2(1.2)	0(0)

a: Significantly different from vehicle control group (p≤0.01)

Conclusions:

TMPCC did not cause any developmental toxicity in the Sprague-Dawley rat at dermal dosages up to 2,000 mg/kg/day.

Executive summary:

The developmental toxicity potential of trimethylolpropane caprylate caproate (TMPCC, CAS no. 11138-60-6) was evaluated in rats. Sprague-Dawley rats were administered TMPCC in a corn oil suspension dermally at dose levels of 0, 200, 600, or 2,000 mg/kg/day on gestation days (GD) 6–15 (sperm positive day5GD 0). Caesarean sections were performed on GD 20 and fetuses were evaluated for viability, growth, and external, visceral, and skeletal abnormalities. Each group consisted of 25 females, with at least 22 per group being pregnant. The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain. There were no differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. TMPCC did not cause any developmental toxicity in the Sprague-Dawley rat at dermal dosages up to 2,000 mg/kg/day.

Reference 5

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Developmental toxicity study with rats, which received a single dose of 18.75 mmol/kg bw octanoic acid by gavage on day 12 of gestation. Fetuses were analysed on day 20 of gestation.

GLP compliance:

no

Limit test:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Octanoic acid from Sigma Chemical Company (St. Louis, MO).
- Analytical purity: no information given

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

Studies were conducted with pregnant Sprague-Dawley rats purchased timemated from Charles River (Portage, MI).
The animals arrived at laboratory on day 6 or 7 of pregnancy, allowing 5 to 6 days for acclimation prior to toxicant administration. Upon arrival, the females were housed in small groups in hanging
wire cages in rooms maintained at a constant temperature (22 ± 1°C), relative humidity (50 ± 5%), under a controlled 12-hr light-dark cycle and fed Purina Rodent Laboratory Chow and water ad libitum.

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Frequency of treatment:

Single dosing

Duration of test:

9 days (from day 12 to day 20 of gestation)

Dose / conc.:

2 700 mg/kg bw/day (nominal)

Remarks:

recalculated dose for 18.75 mmol/kg bw

No. of animals per sex per dose:

Test substance: 12
Control: 10

Control animals:

yes

Details on study design:

Octanoic acid was administered undiluted by oral gavage on the morning of day 12 of rat gestation (day 0 = morning of finding vaginal plug).
On day 20 of gestation, the rats were killed by chloroform overdose. The uterine horns were exposed through incision of the ventral abdominal wall and examined for placement of fetuses and counting of resorption sites. The fetuses were removed from the uterus, examined grossly for the occurrence of malformations, sexed, weighed.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: No:
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined: Uterus
OTHER
- Pharmacokinetics: Content of octanoic acid in maternal plasma at 0.25, 0.5, 1, 2, 4, 8, and 24 h after dosing

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: 1/3 per litter
- Soft tissue examinations: Yes: 2/3 per litter
- Skeletal examinations: Yes: 2/3 per litter
- Head examinations: Yes: 2/3 per litter
-Other: Content of octanoic acid in embryo at 0.25, 0.5, 1, 2, 4, 8, and 24 h after dosing

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Severe not specifies maternal toxicity was noted.

Mortality:

no mortality observed

Body weight and weight changes:

not examined

Dose descriptor:

NOAEL

Effect level:

2 700 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: Dose Level

Fetal body weight changes:

effects observed, non-treatment-related

Details on embryotoxic / teratogenic effects:

Octanoic acid was essentially devoid of embryotoxic effects except for a slight reduction of fetal weight, most likely attributable to the severe maternal toxicity that accompanied the administration of this agent.

Dose descriptor:

NOAEL

Effect level:

2 700 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental toxicity

Abnormalities:

no effects observed

Developmental effects observed:

no

On day 20 of gestation, the rats were killed by chloroform overdose. The uterine horns were exposed through incision of the ventral abdominal wall and examined for

placement of fetuses and counting of resorption sites. The fetuses were removed from the uterus, examined grossly for the occurrence of malformations, sexed, weighed, and then placed in Bouin's fluid (2/3) or in alcohol (1/3). Those in Bouin's fluid were subsequently examined by razor blade sectioning; those in alcohol were double stained for examination of cartilage and bone.

Results on embryotoxicity

	Octanoic acid	Control
Dose (mg/kg bw)	2704	0
No. surv. females/no. treated	11/12	10/10
No. implantation sites	131	108
No. res. or dead (%)	10 (9)	6 (6)
No. surv. malf. (%)	4 (3)	1 (1)
Mean fetal wt., M/F	3.48/3.23	3.90/3.71

Weight of day 20 rat fetuses exposed to an octanoic acid on day 12 of pregnancy:

	Control		Octanoic acid
Litter No.	Male	Female	Male	Female
1	3.6	3.4	3.8	3.7
2	4.2	3.9	3.8	3.5
3	4	3.7	2.5	2.2
4	4	3.9	3.7	3.3
5	4	3.8	3.5	3.2
6	3.9	3.7	3.3	3.2
7	3.5	3.4	3.8	3.7
8	4.3	4.1	4.1	3.9
9	4.5	4.2	2.2	2
10	3.9	3.6	-	-
mean	3.99 ± 0.30	3.77 ± 0.27	3.19 ± 0.65	3.19 ± 0.66
% variability	7.5	7.2	19.1	20.7

Transplacental Pharmacokinetics

Based on the data presented in the publication figures, ca. 70 µg/mL octanoic acid was found in maternal plasma and ca. 200 µg/g fetus directly after administration, respectively. While octanoic acid could not be detected anymore in maternal plasma after ca. 2 h, it took 5 h until octanoic acid could not be detected anymore within the embry

Conclusions:

According to the results, Octanoic acid had very low potential to induce developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 700 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Additional information

Information from supporting substances has been used to fulfil the information requirements for this endpoint.For read across justification, see the attached file in endpoint 13 ( Analogue approach justification):

Oral route

An estimated NOAEL for nonanoic acid of 1820 mg/kg bw and day which was derived from a rat study using MCT (Henwood et al. 1997; MCT = Medium Chain Triglycerides; contain mainly C8 and C10 acid. MCT was also tested in the rabbit (Henwood et al. 1997). Developmental and teratogenicity was seen at the high dose only where the does showed clear maternal toxic effects (significantly reduced feed intake and reduced body weight gain p>0.01). Developmental toxicity and teratogenicity was considered to result from the low feed intake and malnutrition of the does. The NOAEL for nonanoic acid that was derived from this study was therefore only 425 mg/kg bw and day. This value should not be considered for the derivation of a DNEL because the toxic effects were secondary to the low maternal feed intake, rather than to direct toxic effects.

The studies outlined above fulfil the information requirements for the endpoint Developmental Toxicity.

For clarity, the executive summaries of all available studies are presented below.

1 -Time-matedrats(25/group) received intravenous infusions into the caudal vein (duration 4h/day) during gestation Days 6 through 15 of a 20% lipid emulsion that contained a ratio of 3:1 of MCT and Long Chain Triglycerides (LCT) from soy bean oil at doses of 1 and 4.28 g/kg bw and day. Controls received 0.9% saline). Medium chain fatty acids are 6 to 12 carbons in length. The 20% lipid emulsion is composed primarily of 8- and 10-carbon fatty acids, with traces of 6- and 12-carbon fatty acids. The dams were observed for clinical signs and sacrificed at termination. Examinations of the dams and of the uterus content were performed.

No clinical signs or mortalities were noted. Changes in high-dose rats (enlarged lymph nodes, spleen, and renal pelvis; small thymus, small red lung foci) were considered to be related to treatment, hence the NOAEL was 1 000 mg/kg bw and day for maternal toxicity. As to the foetuses, there were no significant differences compared to controls regarding early or late resorptions, post implantation loss, dead or alive foetuses, mean uterine weight and foetal weight (though there was a trend towards lower weights at the high dose), and there were no external, soft tissue or skeletal changes that could be related to treatment. Hence, the NOAEL for developmental toxicity and teratogenicity was 4280 mg/kg bw and day(Henwood et al., 1997).

The study is considered to be valid. The intravenous route was used because the parenteral route is used in patients that cannot meet their nutritional needs by the conventional oral route, and 1000 mg/kg bw and day is the approximate clinical dose, whereas 4280 mg/kg bw and day is reportedly the highest preclinical dose that did not cause narcosis.

The study can be used for the assessment of Carboxylic acids, C5 -9 in a way as this has been described in the repeated dose toxicity section for the studies of Matulka and Webb who also administered triglycerides as test material. In brief,

-   the metabolism of straight chain fatty acids of 8 to 10 carbons including nonanoic acid is very similar and includes rapid degradation via ß-oxidation, which justifies cross reading form C8 and C10-fatty acids to C9 nonanoic acid.

-   The fatty acid composition of the test material of this study (Henwood et al., 1997) was almost exclusively C8 and C10, with traces of C6 and C12.

-   The mean molecular weight of the triglycerides is calculated assuming that traces are negligible, and that C8 and C10 are present in equimolar proportions (this was not specified in the publication). Then, the mean triglyceride molecular weight is 596, and the two fatty acids represent 42.5% each of the molecular weight, and also the dose, i.e. approx. 1820 mg of C8 and also of C10 fatty acid/kg bw and dose.

In a weight of evidence approach, this result can be used to assess the target substance: NOAEL approx.. 1820 mg/kg bw and day.

2- Time-matedrabbits(15/group) received intravenous infusions into the ear vein (duration 5 h/day) during Gestation Days 7 through 19 of a 20% lipid emulsion that contained a ratio of 3:1 of MCT and Long Chain Triglycerides (LCT) from soy bean oil at doses of 1 and 4.28 g/kg bw and day. Controls received 0.9% saline. Medium chain fatty acids are 6 to 12 carbons in length. The 20% lipid emulsion is composed primarily of 8- and 10-carbon fatty acids, with only traces of 6- and 12-carbon fatty acids. The does were observed for clinical signs and sacrificed at termination, the does and of the uterus content were examined.

No treatment-related mortalities were noted. The only clinical sign was a reduced faecal output of 3/15 does on one day. Body weight change and feed intake was significantly reduced (p<0.01) in high-dose rabbits which was expected because of the high caloric test material. No adverse effect was noted in does at 1000 mg/kg bw and day, hence this was considered to represent the maternal NOAEL value.

 

Foetal findings: at the low dose (1000 mg/kg bw and day) foetal postimplantation loss was slightly increased and the mean foetal weight was slightly reduced without gaining a level of significance. This was, however, the case at the high dose where early and late resorptions were clearly increased; consequently, the number of live foetuses was decreased (p<0.01), and the mean foetal weight was significantly decreased (p<0.01). The incidence of external (p<0.05), soft tissue and skeletal malformations (p<0.05) was increased at the high dose.

The incidence of soft tissue findings was also increased in low dose foetuses, but without gaining a level of significance when individual malformations were analysed statistically. Similarly, an increase of skeletal findings was seen in the low dose group below a level of significance. Based on these findings the NOAEL for developmental toxicity was set at 1000 mg/kg bw and day, and the authors assume that the findings at 4280 mg/kg bw and day are attributable to the low feed consumption and malnutrition rather than a direct teratogenic effect of the test material(Henwood et al. 1997).

The study is considered to be valid. The intravenous route was used because the parenteral route is used in patients that cannot meet their nutritional needs by the conventional oral route, and 1000 mg/kg bw and day is the approximate clinical dose, whereas 4280 mg/kg bw and day is reportedly the highest preclinical dose that did not cause narcosis.

The study can be used for the assessment of n-nonanoic acid in a way as this has been described in the repeated dose toxicity section for the studies of Matulka and Webb who also administered triglycerides as test material. In brief,

-   the metabolism of straight chain fatty acids of 8 to 10 carbons is very similar and includes rapid degradation via ß-oxidation.

-    The fatty acid composition of the test material of this study (Henwood et al., 1997) was almost exclusively C8 and C10, with traces of C6 and C12.

-     The mean molecular weight of the triglycerides is calculated assuming that traces are negligible, and that C8 and C10 are present in equimolar proportions (this was not specified in the publication). Then, the mean triglyceride molecular weight is 596, and the two fatty acids represent 42.5% each of the molecular weight, and also of the dose, i.e. approx. 425 mg of C8 and also of C10 fatty acid/kg bw and dose.

In a weight of evidence approach, this result can be used to assess the substance Carboxylic acids C5 -9: NOAEL approx. 425 mg/kg bw and day.

 3. In another study, the substance octaonic acid, a constituent of the target UVCB substance, was tested on rat in terms of the potential to induce developmental toxicity (Scott et al.). According to the results, Octanoic acid had very low potential to induce developmental toxicity, which was clearly due to a low concentration in maternal plasma and very little placental transfer, presumably due to protein binding in maternal plasma (ref. Nau H, Lewandowski C, Klug S, Neubert D. Pharmacokinetic aspects of drug effects in vitro. Toxic in Vitro 2:69-174(1988).) Octanoic acid also possesses low intrinsic activity as shown in rodent whole embryo culture.

4. According to a study of Ambroso et alt., Isooctanoic acid (CAS RN 25103-52-0), a complex isomeric substance containing several aliphatic carboxylic acids, primarily dimethyl hexanoic acid. was tested in terms of potential for developmental toxicity in CD (Sprague-Dawley) rats. It was administered by oral gavage to 25 confirmed-mated females at doses of 0, 200, 400, and 800 mg/kg/day on gestation days (GD) 6-15, based on a range-finding experiment. Maternal body weights, food consumption, and clinical observations were recorded throughout gestation. On GD 21, cesarean sections were performed and the uterine contents removed and subjected to conventional teratological evaluation. At 800 mg/kg/day, maternal body weight gain and food consumption were reduced during the exposure period, and clinical signs were evident. There were no significant differences in fetal weight, malformation incidence, or fetal viability in any of the experimental groups. There was a statistically significant increase in the incidence of total variations in the 800-mg/kg/day group, which was within the historical control range of the laboratory and not considered biologically significant. These results indicate that, isooctanoic acid was not teratogenic or a selective developmental toxicant in rats. This study established a maternal no-observable-adverse-effect level (NOAEL)  at 400 mg/kg/day and a developmental NOAEL at 800 mg/kg/day.

 

According to the literature data (also EFSA opinion 2013), animals and humans are exposed to fatty acids via food feed, fatty acids are rapidly and completely metabolised to carbon dioxide and water, no bioaccumulation has to be considered, and no adverse effects were seen in repeated dose studies even at high doses of MCT, and that no adverse effects of fatty acid ingestion on reproduction is known. Based on this it was concluded that no further developmental toxicity study would be required.

 

 Dermal route:
There is only one study available (Azuka and Daston, 2014) on the read-across substance Trimethylolpropane Caprylate Caprate.

In particular, the developmental toxicity potential of trimethylolpropane caprylate caproate (TMPCC, CAS no. 11138-60-6) was evaluated in rats. Sprague-Dawley rats were administered TMPCC in a corn oil suspension dermally at dose levels of 0, 200, 600, or 2,000 mg/kg/day on gestation days (GD) 6–15 (sperm positive day5GD 0). Caesarean sections were performed on GD 20 and fetuses were evaluated for viability, growth, and external, visceral, and skeletal abnormalities. Each group consisted of 25 females, with at least 22 per group being pregnant. The two highest dose levels caused some local irritation at the site of application, but no decreases in maternal weight gain. There were no differences from control in any of the developmental parameters measured, including embryo/fetal viability, fetal weight, malformations, or variations. TMPCC did not cause any developmental toxicity in the Sprague-Dawley rat at dermal dosages up to 2,000 mg/kg/day.

Justification for classification or non-classification

Based on the results of the combined reprotox / repeated dose toxicity testing and developmental toxicity tests, the test item should not be classified for reproductive effects according to the criteria described in the EU Regulation No. 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).