Fatty acids, tall-oil, reaction products with acrylic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2003 to 11 January 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent, England.
- Age at study initiation: 6 weeks
- Weight at study initiation: Males 154-169g; Females: 149-166g.
- Housing:
Pre-mating: 1/sex/cage, polypropylene cages with stainless steel grid tops and solid bottoms
Mating: 1:1, polypropylene cages with stainless steel grid tops and solid bottoms
Post-mating: female 1/sex/cage, solid-bottom cages with white paper tissue as nesting material

- Diet (e.g. ad libitum): Al libitum, Rat and Mouse Breeder Diet No.3 (Ground) SQC (Special Diets Services Ltd. Stepfield, Witham, Essex, UK)
- Water (e.g. ad libitum): Ad libitum, tap-water
- Acclimation period: 12 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-21
- Humidity (%): 43-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

acetone

Details on exposure:

DIET PREPARATION
- Frequency of preparation of diet: fresh diets were prepared weekly during the study.
- A pre-mix was made by mixing the test item dissolved in acetone with appropriate amounts of Rat and Mouse Breeder Diet No.3 (Ground) SQC (RM3 diet). The high- and intermediate-dose diets were made by mixing appropriate amounts of pre-mix with untreated RM3 diet. The low-dose diet was made by diluting the intermediate-dose diet with appropriate amounts of untreated RM3 diet. The control diet was made using a pre-mix that was made using acetone alone.
- Storage temperature of food: Ambient temperature.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: 7 days in first attempt. If not successful, followed by a second period of 7 days with a male of proven fertility
- Proof of pregnancy: vaginal plug and/or sperm in vaginal smear referred to as day 0 of pregnancy
- No further matings after two unsuccessful attempts.
- After successful mating each pregnant female was caged individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Diet prepared for Week 2 and Week 4 of treatment was sampled. Triplicate samples were withdrawn from each formulated diet, including the Control formulation. The samples were analyzed by Inveresk Toxicology Support Laboratory using a method previously validated in the Inveresk laboratory under a separate protocol and contract (Inveresk Project No. 421274).
Concentrations were within 10% of nominal concentrations and the low coefficient of variation (<2.5%) showed homogeneous distribution of the test item in the diets. The analytical method is not specified in the report.

Duration of treatment / exposure:

Males: 4 weeks overall, starting 2 weeks prior to mating until termination
Females: 2 weeks prior mating, through mating until termination after Day 4 of lactation.

Frequency of treatment:

Continuously via the diet

Details on study schedule:

- The age of the animals at the start of mating was 9-10 weeks.

Remarks:

Doses / Concentrations:
0, 46, 271 and 1324 mg/kg bw/day
Basis:
actual ingested

Remarks:

Doses / Concentrations:
0, 500, 3000 and 15000 ppm
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of a 7-day dose range finding study

Positive control:

Not relevant

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly
- Cage side observations checked included: Posture/condition on first approach (animal undisturbed) checking for: Prostration, Lethargy, Writhing, Circling, Breathing abnormalities, Gait abnormalities, Tremor, Fasciculation, Convultions, Biting (of cage components or self mutilating), Vocalisations, Piloerection. Furthermore, ease of removal from cage, body temperature, condition of the coat, presence of salivation, overall ease of handling

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All the animals were examined for reaction to treatment on each day. The nature, onset and intensity of any signs were recorded.:

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly
Male body weights were recorded once during the week prior to the commencement of treatment and once weekly thereafter until termination.
Female weights were recorded once during the week prior to the commencement of dosing, and weekly thereafter until the start of the mating period, and then on Day 0 of gestation (the day of detection of a positive mating sign) followed by Days 7, 14 and 20 of gestation, and then Days 1 and 4 of lactation (where Day 0 = the day of parturition).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
Water consumption was monitored by visual inspection of the water bottles on a weekly basis throughout the study.

Oestrous cyclicity (parental animals):

Stage of oestrous cycle determined daily in vaginal smears from pairing until mating had occurred and at necropsy.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number of live and dead pups, sex of pups, presence of gross anomalies, examined for the presence of milk in the stomach.
Litters were weighed en masse (by sex) on days 1 and 4 of lactation.

GROSS EXAMINATION OF DEAD PUPS: no

Any deficiencies in lactation or maternal care were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: Following 4 weeks of treatment for males all adult animals were sacrificed by exposure to carbon dioxide, followed by exsanguination.
- Maternal animals: After Day 4 of lactation (Days 5-8 of lactation) for females, all adult animals were sacrificed by exposure to carbon dioxide, followed by exsanguination.

GROSS NECROPSY
All adults were subject to a detailed necropsy under the guidance of a veterinary pathologist. The necropsy consisted of an external and internal
examination. All gross lesions were recorded in terms of location, size, shape, colour, consistency and number.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues were weighed:
Adrenals, brain, epidymides, heart, kidneys, liver, lung, ovaries, pituitary, prostate, spleen, Submaxillary Salivary Gland, testes, thymus, thyroids with Parathyroid, uterus.
The following tissues were prepared for microscopic examination:
Abnormal tissue, adrenals, aorta, brain, epididymides, eyes gastrointestinal tract (stomach, duodenum, jejunum, ileum, caecum, colon, rectum), heart, optic nerve, kidneys, liver, lung, mesenteric lymph node, oesophagus, ovaries, pancreas, pituitary, prostate, sciatic nerve, seminal vesicle, skin+mammary gland, spinal cord, spleen, sternum, submandibular lymph node, Submaxillary Salivary Gland, testes, thigh muscle, thymus, thyroids with Parathyroid, trachea, urinary bladder, uterus, vagina.

Postmortem examinations (offspring):

SACRIFICE
- Pups were sacrificed by injection of sodium pentobarbitone. A random order was used for the necropsy of the adult animals and the pups were sacrificed at the same time as their mother - After day 4 of lactation (day 5-8 of lactation).
- These animals were subjected to postmortem examinations. The pups were examined for externally visible abnormalities

GROSS NECROPSY
- Gross necropsy consisted of external examinations.

HISTOPATHOLOGY / ORGAN WEIGTHS
Not performed.

Statistics:

Selected neurotoxicity, body weight, food consumption, haematology and clinical chemistry data were subjected to analysis of variance or the Kruskal- Wallis non-parametric analysis. Organ weights were analysed as above and by one-way analysis of covariance (ANCOVA) using the terminal kill body weight as covariate. Histological incidence data were analysed using Fisher’s Exact Probability test.
Pairwise comparisons were made using Fisher’s F-protected LSD method via Student’s t-test, or by chi-squared protected z-test (the non-parametric equivalent of Student’s t-test), and were only performed against the Control group.
All statistical tests were two-sided and performed at the 5% significance level.

Reproductive indices:

Fertility Index (male) = Number Siring a litter / Number paired
Fertility Index (female) = Number Pregnant / Number paired
Gestation Index = Number bearing live pups / Number pregnant
Birth Index = Total number of pups born (live and dead) / Number of implantation scars

Offspring viability indices:

Viability Index = Number of pups live on Day 4 of lactation / Number live on Day 0
Live Birth Index = Number of pups live on Day 0 of lactation / Total number born (live and dead)

Clinical signs:

no effects observed

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
All clinical observations and necropsy findings were considered to be consistent with those normally seen in rats of this age and strain.
All clinical signs observed were those commonly noted during this type of study, and there were no significant differences in group incidence or between sexes.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Food consumption was marginally reduced in high-dose males on days 14 and 28.
No obvious treatment-related changes were seen in males or females prior to mating or during gestation and lactation.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The nominal dose levels of 500, 3000 and 15000 p.pm. resulted in mean actual test substance intakes of 46, 271 and 1324 mg/kg bw/day, respectively

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
There were no treatment-related changes in mating preformance. The duration of gestation was similar in all groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
At 15000 p.p.m., there was an increase in liver weight in both sexes. This increase was also evident at 3000 p.p.m., although statistical significance was not achieved at this level. At 15000 p.p.m., there was a slight but significant increase in the absolute weight of kidneys in females.
At 15000 p.p.m. in females, there was a decrease in spleen weight. A marginal reduction in the spleen weight in high-dose males did not reach statistical significance. There was a dose related reduction in absolute thymus weights in females which attained statistical significance at 15000 p.p.m. The absolute thymus weights in low- and intermediate-dose females were not statistically significantly different from Control.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no obvious intergroup differences in either sex.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histological examination of the liver revealed centrilobular hepatocyte hypertrophy at 15000 p.p.m. in 2/5 males and 4/4 females examined.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased liver weights and increased centrilobular hepatocyte hypertrophy in males and females in the highest dose

Clinical signs:

no effects observed

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:

not examined

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
There was no obvious effect of treatment on the mean number of implants, live young per litter or on litter survival as indicated by the birth index and
viability index.

BODY WEIGHT (OFFSPRING)
At 15000 p.p.m, and to a lesser extent at 3000 p.p.m., there was a slightly reduced mean pup weight in males and females, reflecting the slightly larger number of live pups per litter at these levels. Group mean litter weights at both levels were comparable with Control.
Group mean litter and pup weights at 500 p.p.m. were comparable to Control.

GROSS PATHOLOGY (OFFSPRING)
Only minor findings such as pups cold and/or scattered, that are typical for pre-weanings were observed but have not been reported, these findings are retained in the study data.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 324 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There were no treatment-related changes in reproductive parameters in any of the dose groups.

Reproductive effects observed:

not specified

Not applicable

Conclusions:

Toxicity to reproduction/developmental toxicity potential of Diacid 1550 was investigated in a combined 28-d repeated dose toxicity/reproscreening study that was performed in accordance with OECD422 and under GLP conditions. Diacid 1550 was administered orally via the diet at 0, 500, 3000, and 15000 ppm. There were no treatment-related changes observed in reproductive parameters in any of the dose groups. Therefore, the NOAEL for reproductive parameters was considered 15000 p.p.m. (corresponding to 1324 mg/kg bw/day), under the conditions of this study.

Executive summary:

This study was designed to provide initial information on possible effects on reproduction and/or development of Diacid 1550 in the rat after oral administration via the diet for at least weeks. The study was performed in accordance with OECD 422 and under GLP conditions.

 

Four groups of 10 male and 10 female Sprague-Dawley rats received Diacid 1550 itemviathe diet at a constant concentration of 0, 500, 3000 and 15000 p.p.m (corresponding to 0, 46, 271 and 1324 mg/kg bw/day). The males were treated for 2 weeks prior to mating, through until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation until at least Day 4 of lactation. The animals were monitored daily for any signs of ill health or reaction to treatment. Detailed functional observations were performed weekly, with additional functional observations performed on 5 males and 5 females per group on two occasions (pre-trial and in week 4 for males and during lactation for females). Blood samples were also taken from 5 males and 5 females per group for laboratory investigations. All adult animals were killed and subjected to a detailed necropsy examination after completion of treatment. Representative tissue samples were taken from all adults and a selection of tissues were weighed. Histopathology was conducted on tissues from 5 rats/sex from Control and High dose groups; the same animals that were used for laboratory investigations.Pups were examined externally.

 

At 15000 p.p.m., there was an increase in liver weights that was associated with an increase in centrilobular hepatocyte hypertrophy which was observed in 2/5 males and 4/5 females examined. Changes in liver function were demonstrated with an increase in alkaline phosphatase. Clinical chemistry at this level also revealed an increase in albumin in both sexes, a reduction in cholesterol and an increase in phosphate in females. Furthermore, in females kidney weight was markedly increased and spleen weight was markedly decreased. A decrease in thymus weights of females was also evident at this level.

At 3000 p.p.m., liver weight was also increased, although not statistically significant. An increase in the levels of alkaline phosphatase was also seen in both sexes. In females, there was an increase in albumin and a slight increase in phosphate, as well as a reduction in cholesterol. At 500 p.p.m. there was an increase in alkaline phosphatase in males, and reduced cholesterol in females. The reduction in cholesterol was dose-related. As the values are all within the historical ranges in Sprague-Dawley rats, they were considered not adverse (Jung-Min Lee et al., Laboratory Animal Research, 2012: 28(2), 115-121).

There were no obvious effects of treatment noted during neurotoxicity observations or on the mating performance.

 

There was no obvious effect of treatment on the mean number of implants, live young per litter or on litter survival as indicated by the birth index and viability index. At 15000 p.p.m, and to a lesser extent at 3000 p.p.m., there was a slightly reduced mean pup weight in males and females, reflecting the slightly larger number of live pups per litter at these levels. Group mean litter weights at both levels were comparable with Control. Group mean litter and pup weights at 500 p.p.m. were comparable to Control. Only minor findings such as pups cold and/or scattered, that are typical for pre-weanings were observed but have not been reported, these findings are retained in the study data.

 

In conclusion, based on increased liver weights and increased centrilobular hepatocyte hypertrophy in males and females at the highest does, the parental NOAEL was considered to be 3000 p.p.m., corresponding to 271 mg/kg bw/day, under the conditions of this study.. For reproductive parameters the NOEL was considered to be 15000 p.p.m., corresponding to 1324 mg/kg bw/day.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 324 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

This study was designed to provide initial information on possible effects on reproduction and/or development of Diacid 1550 in the rat after oral administration via the diet for at least weeks. The study was performed in accordance with OECD 422 and under GLP conditions.

Four groups of 10 male and 10 female Sprague-Dawley rats received Diacid 1550 itemviathe diet at a constant concentration of 0, 500, 3000 and 15000 p.p.m (corresponding to 0, 46, 271 and 1324 mg/kg bw/day). The males were treated for 2 weeks prior to mating, through until necropsy after at least 4 weeks of treatment. Females were treated for 2 weeks prior to mating, then through mating, gestation until at least Day 4 of lactation. The animals were monitored daily for any signs of ill health or reaction to treatment. Detailed functional observations were performed weekly, with additional functional observations performed on 5 males and 5 females per group on two occasions (pre-trial and in week 4 for males and during lactation for females). Blood samples were also taken from 5 males and 5 females per group for laboratory investigations. All adult animals were killed and subjected to a detailed necropsy examination after completion of treatment. Representative tissue samples were taken from all adults and a selection of tissues were weighed. Histopathology was conducted on tissues from 5 rats/sex from Control and High dose groups; the same animals that were used for laboratory investigations.Pups were examined externally.

At 15000 p.p.m., there was an increase in liver weights that was associated with an increase in centrilobular hepatocyte hypertrophy which was observed in 2/5 males and 4/5 females examined. Changes in liver function were demonstrated with an increase in alkaline phosphatase. Clinical chemistry at this level also revealed an increase in albumin in both sexes, a reduction in cholesterol and an increase in phosphate in females. Furthermore, in females kidney weight was markedly increased and spleen weight was markedly decreased. A decrease in thymus weights of females was also evident at this level.

At 3000 p.p.m., liver weight was also increased, although not statistically significant. An increase in the levels of alkaline phosphatase was also seen in both sexes. In females, there was an increase in albumin and a slight increase in phosphate, as well as a reduction in cholesterol. At 500 p.p.m. there was an increase in alkaline phosphatase in males, and reduced cholesterol in females. The reduction in cholesterol was dose-related. As the values are all within the historical ranges in Sprague-Dawley rats, they were considered not adverse (Jung-Min Lee et al., Laboratory Animal Research, 2012: 28(2), 115-121).

There were no obvious effects of treatment noted during neurotoxicity observations or on the mating performance.

There was no obvious effect of treatment on the mean number of implants, live young per litter or on litter survival as indicated by the birth index and viability index. At 15000 p.p.m, and to a lesser extent at 3000 p.p.m., there was a slightly reduced mean pup weight in males and females, reflecting the slightly larger number of live pups per litter at these levels. Group mean litter weights at both levels were comparable with Control. Group mean litter and pup weights at 500 p.p.m. were comparable to Control. Only minor findings such as pups cold and/or scattered, that are typical for pre-weanings were observed but have not been reported, these findings are retained in the study data.

In conclusion, based on increased liver weights and increased centrilobular hepatocyte hypertrophy in males and females at the highest does, the parental NOAEL was considered to be 3000 p.p.m., corresponding to 271 mg/kg bw/day, under the conditions of this study.. For reproductive parameters the NOEL was considered to be 15000 p.p.m., corresponding to 1324 mg/kg bw/day.

Short description of key information:
Reproscreening study (OECD422): No effects observed on development or reproduction, NOAEL 1324 mg/kg bw/d (oral, rat)

Justification for selection of Effect on fertility via oral route:
The study is the only available study with regard to developmental toxicity/toxicity to reproduction and was performed according to OECD422 and under GLP-conditions.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In the reproscreening study no effects on development were observed.

Justification for classification or non-classification

Based on the available information from the reproscreening study, Diacid 1550 does not have to be classified ar reprotoxic/toxic to the development in accordance with the criteria outlined in Annex VI of 67/548/EEC and Annex I of 1272/2002/EEC.