Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to actual guidelines conducted under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

Molecular formula:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

IUPAC Name:

Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): Octadecanoic acid, 9, 10-dihydroxy,
monoammonium salt due to composition renamed to Reaction product of fatty acids C14-18/C16-18 unsaturated with dihydrogendioxide and Ammonia
- Molecular formula (if other than submission substance): C18H36O4H3N
- Molecular weight (if other than submission substance):333 g/mol
- Physical state: Viscous liquid, yellowish
- Analytical purity: see analytical report
- Composition of test material, percentage of components: purity approximately 50% in water
- Lot/batch No.: 0008050156
- Expiration date of the lot/batch:18 January 2013
- Storage condition of test material: ambient temperature
- Other: A report describing the characterization of the test item is included in Annex 4 (study 12L00047).

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RccHan: WIST strain, Harlan, Horst, The Netherlands
- Age at study initiation: 11-12 weeks
- Weight at study initiation: initial body weight variation did not exceed ±20% of the mean weight for each sex
- Housing: Animals were housed in macrolon cages with a bedding of wood shavings (Lignocel, Type ¾) and strips of paper (Enviro-dri) or a wooden block as environmental enrichment
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24°C (24.4ºC in one occasion during room cleaning)
- Humidity (%): relative humidity of at least 45% and not exceeding 65%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: tap water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dilutions of the test substance in the vehicle and the vehicle for dosing the controls were
prepared weekly (namely 27 April, 2, 10, 16, 23 and 30 May, 6 and 13 June 2012), and stored
in a refrigerator (2-10ºC), in portions sufficient for one day. A dosing volume of
5 mL/kg body weight was used for all groups. Dosing volume was adjusted based on the
latest body weight.

VEHICLE
- Amount of vehicle (if gavage): 5mL/kg bw
- Purity: tap water

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: Animals were caged together until mating occurred or 1 week had elapsed.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Sperm positive females that turn out to be non-pregnant were killed not earlier than 27 days after copulation.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): Upon evidence of copulation the females were caged individually for the birth and rearing of their pups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

see annex V of report
Concentration was determined in samples from the batches prepared on 02 May 2012 and 30 May 2012. Homogeneity was determined in samples from 30 May 2012. The concentrations were determined according to a method validated in TNO Triskelion study 20139/01.

Duration of treatment / exposure:

Male animals were dosed during a 2-week premating period, during mating and up to the day of sacrifice after the mating period. The female animals were dosed during a 2-week premating period, and during mating, gestation and lactation up to the day of sacrifice (approx. day 4 of lactation).

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200mg/kg bw
Basis:
nominal conc.
(a.i. 50%, 100mg/kg bw a.i. )

Doses / concentrations 2

Remarks:

Doses / Concentrations:
600mg/kg bw
Basis:
nominal conc.
(a.i. 50%, 300mg/kg bw a.i. )

Doses / concentrations 3

Remarks:

Doses / Concentrations:
1000mg/kg bw
Basis:
nominal conc.
(a.i. 50%, 500mg/kg bw a.i. )

No. of animals per sex per dose:

12

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: During 1-4 May 2012, the animals in the high-dose group received 2000 mg/kg (a.i. 1000mg/kg) body weight. Based on unscheduled deaths (females 81 and 87) and clinical signs the high-dose level was reduced to 1000 mg/kg body weight from 5
May 2012.

Positive control:

none

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily in the morning and afternoon
- Cage side observations checked in table were included: All abnormalities, signs of ill health or reactions to treatment were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily in the morning

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded shortly before the start of
administration of the test item and at the start of the study. Males were weighed weekly until sacrifice Females were weighed once per week during the premating period. Mated females were weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation. Non-mated females were weighed once per week after the mating period. In addition, the animals were weighed on their scheduled necropsy date.

OTHER:
Food consumption
Food consumption was measured per cage over weekly intervals during the study, except for the mating period, during which no food consumption was registered.

Litter observations:

At the end of the gestation period (GD 21), females were examined twice daily for signs of parturition. Any difficulties occurring during parturition were recorded. To keep nest disturbance to a minimum the litters were examined only once daily for dead pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- total litter size and numbers of each sex as well as the number of stillbirths, live- and dead pups and grossly malformed pups was evaluated on days 0 and 4 of lactation.
- pups were individually weighed on days 0 and 4 of lactation.

GROSS EXAMINATION OF DEAD PUPS:
yes, Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded. At necropsy of the dams, at day 4 of lactation, pups were examined externally for gross abnormalities and killed by freezing under CO2/O2 anaesthesia.

Postmortem examinations (parental animals):

SACRIFICE
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was also performed on animals that died or were killed intercurrently.

GROSS NECROPSY
- Gross necropsy performed

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated below were prepared for microscopic examination and weighed, respectively.
- ovaries (after counting the corpora lutea)
- oviduct
- uterus (after counting of the implantation sites, including cervix)
- vagina
- testes
- coagulation gland
- epididymes,
- seminal vesicles (including coagulation gland)
- prostate
- all gross lesions

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at day 4 of lactation.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows: Grossly malformed pups were sacrificed and examined. A necropsy was performed on stillborn pups and pups dying during the study; macroscopic abnormalities were recorded

GROSS NECROPSY
- Gross necropsy performed

see litter observation

Statistics:

Continuous data were subjected to a decision tree or parametric statistical test.
Dichotomous data were evaluated using the statistical test Chi-square-Fisher.

Tests were generally performed as twosided tests with results taken as significant where the probability of the results is p<0.05 (*) or p<0.01 (**).

Reproductive indices:

The following parameters were calculated:
- pre-coital time = time between the start of mating and successful
copulation
- duration of gestation = time between gestation day 0 and day of
delivery
- female mating index = (number of females inseminated/number of females
placed with males) x 100
- male fertility index = number of males with pregnant females*100/number of
males placed with females
- female fertility index = number of pregnant females*100/number of inseminated
females
- gestation index = (number of females with live pups / number of
females pregnant) x 100
- live birth index = (number of pups born alive/number of pups born)
x 100
- viability index day 0 - 4 = (number of pup surviving 4 days/number of
liveborn on day 0) x100
- pup mortality day 0 or 4 = (number of dead pups on day 0 or 4/ total number
of pups on day 0 or 4) x 100
- sex ratio day 0 or 4 = (number of live male fetuses or pups on day 0 or
4/ number of live fetuses or pups on day 0 or 4)
x 100
- pre-implantation loss = [(number of corpora lutea – number of implantation sites)/number of
corpora lutea x 100
- number of los implantations = number of implantation sites – number of pups born alive
- post-implantation loss = [(number of implantation sites - number of pups
born alive)/ number of implantation sites] x 100.

Offspring viability indices:

see reproductive indices

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

grunting respiration, mainly traced back to treatment methode (gavage)

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

At the beginning of the study three females, which were found dead or killed in extremis (3-55, 4-81 and 4-87) were replaced by reserve rats. Taking into account the macroscopic and microscopic changes observed, it was assumed that the mortality was due regurgitation of irritating test item. Therefore, the observed mortality was ascribed to the dosing procedure (oral gavage) rather than to systemic toxicity of the test substance.

Clincal signs during experiment:
Two females of the high-dose group died. female 79 was found dead on day 13, female 95 was killed moribund on day 20. Several male and female rats showed a grunting respiration during the study. This was observed in 2 control males, 2 low-dose males, 5 mid-dose males + 4 mid-dose females and in 3 high-dose males + 6 high-dose females. This response generally occurred only occasionally during the premating, mating, gestation or the lactation period. In three rats (mid-dose female 55, high-dose male 80 and high-dose female 93) grunting respiration occurred more consistently during several days. This observation is considered to be related to the treatment method (oral gavage).

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

No treatment-related clinical signs were observed during lactation.

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic examination of the stillborn pup in the low-dose group showed no abnormalities for this pup

Histopathological findings:

not examined

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the absence of any signs of systemic toxicity in surviving rats, the No Observed
Adverse Effect Level (NOAEL) for parental toxicity is considered to be 1000 mg/kg bw/day test item (a.i. 500mg/kg bw/day). No treatment-related effects were observed on fertility and developmental parameters. Therefore, the NOAEL for fertility and development is established at the high-dose of 1000 mg/kg bw/day test item (a.i. 500mg/kg bw/day).