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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

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

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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

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 - 169 g; Females: 149 - 166 g.
- 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): Ad 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 oral 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 inrermediate-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.

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 using a previously validated method. 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

Dose / conc.:

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

Remarks:

negative control

Dose / conc.:

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

Remarks:

equivalent to 500 ppm in diet

Dose / conc.:

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

Remarks:

equivalent to 3000 ppm in diet

Dose / conc.:

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

Remarks:

equivalent to 15000 ppm in diet

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
A one-week dose range finding study was performed in which groups of 5 male and 5 female rats were exposed to 0, 1000, 5000, and 20000 ppm via the diet. Pathology findings revealed a dose related increase in liver weight in females at all levels, and an increase in liver weight in males at 20000 ppm. Whilst these findings were considered most likely to represent an adaptive response it was considered appropriate to use lower levels in the main study. In addition, at 5000 and 20000 ppm, there was a decrease in spleen weight. From the findings in this study dose levels of 500, 3000 and 15000 ppm were considered appropriate for use in the main study.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Weekly
- Cage side observations:
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.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Condition of the eyes was checked for pupillary function (reaction to visual stimulus), miosis, mydriasis, exophthalmos, encrustation, and lacrimation
- Time schedule for examinations: Weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 4 of treatment for males and on Day 5/6 of lactation for females
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: Blood samples were obtained from 5 males and 5 females from each dose group. 40 animals.
For males, the first 5 animals in each group were tested. For females, the first 5 animals to have reared their litter to Day 5/6 of lactation were tested.

- Parameters examined:
Haemaglobin
Red Blood Cell Count
Haematocrit
White Blood Cell Count
Mean Cell Volume
Mean Cell Haemoglobin
Mean Cell Haemaglobin Concentration
Platelets
Differential White Blood Cell Count:
*Neutrophils
*Lymphocytes
*Monocytes
*Eosinophils
*Basophils
*Large Unclassified Cells

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 4 of treatment for males and on Day 5/6 of lactation for females
- Animals fasted: No
- How many animals: Blood samples were obtained from 5 males and 5 females from each dose group.
- Parameters checked:
Urea
Glucose
Aspartate Aminotransferase
Alanine Aminotransferase
Sodium
Potassium
Chloride
Total Protein
Albumin
AG Ratio
Creatinine
Calcium
Phosphate
Total Bilirubin
Cholesterol
Alkaline Phosphatase

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: All the groups
- Battery of functions tested: sensory activity / grip strength / motor activity / behavioural assessment

OTHER: Coagulation.
- Parameters checked:
Prothrombin Time

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, full external examination, macroscopic abnormalities were recorded

ORGAN WEIGHTS: Yes, recorded for: Adrenals, brain, epidymides, heart, kidneys, liver, lung, ovaries, pituitary, prostate, spleen, Submaxillary Salivary Gland, testes, thymus, thyroids with Parathyroid, uterus.

HISTOPATHOLOGY: Yes, see below for which tissues
RESPIRATORY SYSTEM
-TRACHEA
-LUNG
HAEMOPOIETIC SYSTEM
-LYMPH NODE (MANDIBULAR)
-LYMPH NODE (MESENTERIC)
-SPLEEN
-THYMUS
CARDIOVASCULAR SYSTEM
-HEART
-AORTA
ENDOCRINE SYSTEM
-THYROID GLAND
- PARATHYROID GLAND
- ADRENAL GLAND
- PITUITARY GLAND
- PANCREAS (ENDOCRINE)
GENITAL SYSTEM
- TESTIS
- PROSTATE
- SEMINAL VESICLE
- OVARY
- UTERUS
- VAGINA
URINARY SYSTEM
- KIDNEY
- URINARY BLADDER
ALIMENTARY SYSTEM
- OESOPHAGUS
- STOMACH
- DUODENUM
- JEJUNUM
- ILEUM
- CAECUM
- COLON
- RECTUM
- LIVER
- SALIVARY GLAND (SUBMAXILLARY)
- PANCREAS (EXOCRINE)
INTEGUMENTARY SYSTEM
- SKIN AND SUBCUTIS
- MAMMARY GLAND
NERVOUS SYSTEM
- EYE
- OPTIC NERVE
- BRAIN
- SPINAL CORD
- SCIATIC NERVE
MUSCULO SKELETAL SYSTEM
- SKELETAL MUSCLE
- STERNUM

Other examinations:

Not relevant

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.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

FOOD CONSUMPTION
In males treated at 15000 ppm, there was a marginal reduction in food consumption on Days 14 and 28 of treatment. In females, there were no obvious effects of treatment on food consumption, slight intergroup differences were considered to be incidental.

BODY WEIGHT
In males treated at 15000 ppm, there was a slight reduction in body weight gain on commencement of treatment which persisted throughout the treatment period. The mean body weights were not significantly different from control. There were no obvious effects of treatment in males treated at 500 or 3000 ppm, or in any of the treated females prior to mating or during gestation and lactation.

CLINICAL CHEMISTRY
In all treated males, and females at 3000 and 15000 ppm, there was a statistically significant increase in alkaline phosphatase.
At 3000 and 15000 ppm in females and 15000 ppm in males, there was a statistically significant increase in albumin. There was also a significant increase in the albumin globulin ratio in females treated at 15000 ppm and a decreased globulin level. There was an increase in the albumin globulin ratio in the males also but this did not achieve statistical significance. In treated females, there was a dose related reduction in cholesterol. A similar reduction could be seen in males treated at 15000 ppm, although this did not achieve statistical significance.
At 15000 ppm in females, there was a marginal increase in phosphate (P <0.05), there was also an increase at 3000 ppm although this did not achieve statistical significance. Other intergroup differences were considered to be incidental and not related to treatment with Diacid 1550.
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).

ORGAN WEIGHTS
At 15000 ppm, there was an increase in liver weight in both sexes. This increase was also evident at 3000 ppm, although statistical significance was not achieved at this level. At 15000 ppm, there was a slight but significant increase in the absolute weight of kidneys in females.
At 15000 ppm 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 ppm. The absolute thymus weights in low- and intermediate-dose females were not statistically significantly different from control.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histological examination of the liver revealed centrilobular hepatocyte hypertrophy at 15000 ppm in 2/5 males and 4/4 females examined.

The observed increase in liver weights was associated with an increase in centrilobular hepatocyte hypertrophy in the high-dose group. Changes in liver function was demonstrated with an increase in alkaline phosphatase. This increase in alkaline phosphatase was seen in all treated males and in mid- and high-dose females. Based on these treatment-related changes in all dose groups a NOEL cannot be established. The LOEL is set at 500 ppm, corresponding to a dietary intake of 46 mg/kg bw/day.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

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

Critical effects observed:

not specified

Conclusions:

The repeated dose toxicity 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. 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 ppm, corresponding to 271 mg/kg bw/day, under the conditions of this study.

Executive summary:

This study was designed to assess the repeated dose toxicity and neurotoxicity of Diacid 1550 in the rat after oral administration via the diet for at least 4 weeks, and to provide initial information on possible effects on reproduction and/or development. 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 item via the diet at a constant concentration of 0, 500, 3000 and 15000 ppm (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 ppm, 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 ppm, 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 ppm 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.

 

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 ppm, corresponding to 271 mg/kg bw/day, under the conditions of this study.

Cross-reference 2

Reason / purpose for cross-reference:

reference to same study

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

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 - 169 g; Females: 149 - 166 g.
- 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.

Dose / conc.:

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

Remarks:

negative control

Dose / conc.:

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

Remarks:

equivalent to 500 ppm in diet

Dose / conc.:

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

Remarks:

equivalent to 3000 ppm in diet

Dose / conc.:

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

Remarks:

equivalent to 15000 ppm 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 ppm 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 ppm, there was an increase in liver weight in both sexes. This increase was also evident at 3000 ppm., although statistical significance was not achieved at this level. At 15000 ppm, there was a slight but significant increase in the absolute weight of kidneys in females.
At 15000 ppm 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 ppm. 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 ppm 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 ppm, and to a lesser extent at 3000 ppm, 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 ppm 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 ppm (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 item via the diet at a constant concentration of 0, 500, 3000 and 15000 ppm (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 ppm, 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 ppm, 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 ppm 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 ppm, and to a lesser extent at 3000 ppm, 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 ppm 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 ppm, corresponding to 271 mg/kg bw/day, under the conditions of this study. For reproductive parameters the NOEL was considered to be 15000 ppm, corresponding to 1324 mg/kg bw/day.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 422 (Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diacid 1550

IUPAC Name:

Diacid 1550

Test animals

Species:

rat

Strain:

Sprague-Dawley

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 - 169 g; Females: 149 - 166 g.
- 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

Administration / exposure

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.

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.

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.

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

control

Doses / concentrations 2

Dose / conc.:

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

Remarks:

equivalent to 500 ppm in diet

Doses / concentrations 3

Dose / conc.:

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

Remarks:

equivalent to 3000 ppm in diet

Doses / concentrations 4

Dose / conc.:

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

Remarks:

equivalent to 15000 ppm in diet

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

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

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes /
DETAILED CLINICAL OBSERVATIONS: Yes
BODY WEIGHT: Yes
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes

POST-MORTEM EXAMINATIONS: Yes

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: No data

Fetal examinations:

-Viability
- Body weight
- Gross pathology

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.

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)

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Details on results:

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 ppm 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 ppm, there was an increase in liver weight in both sexes. This increase was also evident at 3000 ppm, although statistical significance was not achieved at this level. At 15000 ppm, there was a slight but significant increase in the absolute weight of kidneys in females.
At 15000 ppm 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 ppm. 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 ppm in 2/5 males and 4/4 females examined.

Effect levels (maternal animals)

Dose descriptor:

NOAEL

Effect level:

271 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

gross pathology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 15000 ppm, and to a lesser extent at 3000 ppm, 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 ppm were comparable to control.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

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

Other effects:

no effects observed

Description (incidence and severity):

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.

Effect levels (fetuses)

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reduction in number of live offspring

fetal/pup body weight changes

Fetal abnormalities

Abnormalities:

no effects observed

Overall developmental toxicity

Developmental effects observed:

no

Applicant's summary and conclusion

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 ppm (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 ppm (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 ppm, 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 ppm, 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 ppm 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 ppm, and to a lesser extent at 3000 ppm, 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 ppm 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 ppm, corresponding to 271 mg/kg bw/day, under the conditions of this study. For reproductive parameters the NOEL was considered to be 15000 ppm, corresponding to 1324 mg/kg bw/day.