Fatty acids, C18-unsatd., dimers, oligomeric reaction products with tall-oil fatty acids and triethylenetetramine

Repeated dose toxicity: oral

Administrative data

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:

12 June 2012 to 01 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to relevant testing guidelines. A Klimisch score of 2 is assigned, as analytical verification of test concentrations was not possible due to low recoveries.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

analytical verification of test concentrations was not possible due to low recoveries.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

TETA – Fatty acids adducts (Mw 600-1000Da)

IUPAC Name:

TETA – Fatty acids adducts (Mw 600-1000Da)

Constituent 2

Reference substance name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

High molecular weight adducts of Fatty acids, C18-unsatd dimers and trimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 3

Reference substance name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

IUPAC Name:

lower molecular weight adducts of Fatty acids, C18-unsatd dimers with amines, polyethylenepoly-, triethylenetetramine fraction

Constituent 4

Reference substance name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

EC Number:

292-587-7

EC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Cas Number:

90640-66-7

IUPAC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): TOFA_DimerFA_TETA_PAA
- Physical state: Yellow liquid with a brown hue.
- Analytical purity: 100%
- Lot/batch No.: BB001030V1
- Expiration date of the lot/batch: 30 May 2013
- Storage condition of test material: When not in use the test article was stored in a sealed container, at room temperature in the dark.

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were healthy male and female Crl:WI(Han) rats, obtained from Charles River (UK) Ltd., Margate, UK. The range-finding animals were approximately 10-12 weeks of age and weighed 293.3 to 361.1 g (males) and 189.9 to 206.4 g (females) at the start of dosing. The main study animals were approximately 10-12 weeks of age and weighed 293.5 to 348.0 g (males) and 175.7 to 219.5 g (females) at the start of dosing.
The animals were housed in a single, exclusive room, air-conditioned to provide 15 to 20 air changes/hour and maintained at a temperature of 20 to 24°C and a relative humidity of 45 to 65%. Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours darkness. The animals were housed in groups of three during the range-finding phase. Main study animals were housed in groups of up to four (pre-pairing and post pairing), one female with one male (pairing) and the females were housed individually once mated. In addition, relevant animals were housed individually for approximately 24 hours prior to FOB assessment.
SQC Rat and Mouse Breeder Diet No 3, Expanded, (Special Diets Services Ltd, Witham) and mains water was provided ad libitum.
All animals were given an inspection for ill health on arrival, and acclimatised for 15 days.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Formulations were prepared daily as a suspension in corn oil, and administered by gavage. Dose volumes were 5 mL/kg; individual dose volumes were based on individual body weights. Formulations were stored at room temperature in a sealed container, and were stirred continuously before and throughout dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On Days 1 and 8 of dosing in the range-finding study, two aliquots were taken at random from the control/vehicle formulations and three aliquots were taken from the top and bottom of the test material formulation. Samples were stored at <-10°C pending possible analysis. On Days 1, 22 and 42 of the main study, two aliquots were taken at random from the control/vehicle formulations and three aliquots were taken from the top and bottom of the test material formulation. Samples were stored at <-10°C and dispatched to Covance Laboratories Inc., Madison, Wisconsin for analysis.

Duration of treatment / exposure:

Range-finding: 14 days
Main study: Males were dosed daily for 2 weeks prior to pairing, during the pairing period and a further 2 weeks before necropsy; a total of 6 weeks treatment prior to necropsy. Females were dosed once daily for 2 weeks prior to pairing, during the pairing period and until Day 4 post-partum inclusive (7 weeks prior to necropsy). The females were allowed to litter and rear their offspring to Day 4 post-partum. Dosing was deferred or omitted if the dam was in or near parturition.

Frequency of treatment:

Once daily.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
other: nominal, range-finding study

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw/d
Basis:
other: nominal, main study

No. of animals per sex per dose:

Range-finding: 3/sex/dose
Main study: 10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Animals were assigned to treatment groups on arrival using a total randomisation procedure.
Dose levels for the range-finding study were based on the results of the acute oral toxicity study in female rats, in which there were no deaths or clinical signs of toxicity following a single oral dose of 2000 mg/kg bw. During the range-finding study, daily administration was generally well-tolerated with no remarkable clinical signs at any dose level. Mean body weight gain and food consumption were reduced at 300 and 1000 mg/kg bw/d. There were no remarkable findings at necropsy and no adverse effects on haematology or clinical chemistry. Based on these findings, the same dose levels were considered suitable for the main phase.
For the mating procedure please refer to Section 7.8.1.

Positive control:

Not required.

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS
All animals were observed twice daily for general health. All animals were observed daily for signs of ill health or overt toxicity. In addition, the clinical condition of each animals was recorded on days of body weight recording.
All animals were observed post-dosing upon return to their home cage, and at 0.5, 1, 2 and 4 hours post-dose.

BODY WEIGHT
During the range-finding study body weights were recorded on Day -7, Days 1, 4, 7, 10, 13 and on the day of necropsy. During the main study males were weighed on Day -7, then at weekly intervals and the day prior to necropsy. During the main study females were weighed on Day -7, weekly prior to pairing and until confirmation of mating, on Days 0, 7, 14 and 20 of gestation, on Days 1 and 4 post-partum and on the day of necropsy.

FOOD CONSUMPTION
During the range finding study food consumption was recorded over the same intervals as the body weights. During the main study food consumption was also recorded over the same intervals as the body weights for all males prior to and post pairing and females prior to pairing. In addition, food consumption was determined for females over Days 0 to 4, 4 to 7, 7 to 10, 10 to 14, 14 to 17 and 17 to 20 of gestation and Day 1 to 4 post-partum.

URINALYSIS
Urine samples were collected overnight from 5 males/group in Week 7. Food and water were removed during collection. The following parameters were determined: volume (measured by weight, reported in mL (1 g considered equivalent to 1 mL), colour, turbidity, specific gravity, pH#, protein#, glucose#, ketones#, urobilinogen#, bilirubin#, blood#, microscopy. # determined semi-quanitatively.

CLINICAL CHEMISTRY & HAEMATOLOGY
Blood samples were collected from all range-finding phase animals on the day of necropsy. In the main study, blood samples were collected from from five males/group in Week 7, and five females/group on Day 5 post-partum. All samples were collected after an overnight period without food, immediately prior to necropsy.
The following haematology parameters were determined on blood (0.5 mL nominal) taken into EDTA anticoagulant: haemoglobin concentration, haemoglobin distribution width, red blood cell count, red cell distribution width, packed cell volume platelet count (includes platelet clump assessment - clump count below 100 considered as none detected; clump count over 100 considered as platelet clumps present and confirmed by review of Advia cytogram or blood film examination), Reticulocytes, platelet crit, mean cell volume, mean platelet volume, mean cell haemoglobin, platelet distribution width, mean cell haemoglobin concentration, total and differential white cell count.
The following clinical chemistry parameters were determined on plasma derived from whole blood (0.6 mL nominal) collected into lithium heparin anticoagulant: aspartate aminotransferase, albumin, alanine aminotransferase, globulin, alkaline phosphatise, albumin/globulin ratio, sodium, total cholesterol, potassium, glucose, calcium, urea, inorganic phosphorus, total bilirubin, chloride, creatinine, total protein.

BEHAVIOURAL EXAMINATION
All main study animals were subjected to a battery of behavioural tests and observations (functional observation battery; FOB) before initiation of treatment and at once weekly intervals thereafter. Observers were blinded to treatment group, with the exception of Day 4 post-partum functional tests because the protocol specified that the first 5 littered females/group were to be observed the observer had to know which animals to observe to ensure none were missed in error. As the tests were not open to personal interpretation this had no effect on the integrity of the data. Before removal from the cage, each animal was observed and evaluated for the following: posture, activity, gate, tremor, convulsion, excessive vocalisation, arousal upon opening cage. Each animal was then removed from its cage and observed for the following: ease of removal, ease of handling, excessive vocalisation, tremor, convulsion, palpebral closure, exopthalmus, lacrimation, lacrimation type, salivation, respiration, piloerection, appearance of fur, other.
Each main study animal was placed into an open field arena for two minutes, once weekly. Animals were observed for the following: latency to first step, posture, arousal, circling, gait type, gait type severity, stereotypy, tremor, convulsion, other. The number of rears, faecal boli and urine pools, faecal consistency and the presence of polyuria were also recorded. During the last week of dosing (Day 4 post-partum), five males per group and the first five littered females per groups were assessed for auditory startle response, forelimb grip strength, visual placing response and hindlimb grip strength. Locomotor activity of each main study animal was assessed in an automated photocell activity recorder for 30 minutes in the last week of dosing (five study males per group) and on Day 4 post-partum (the five first littered females per group). Activity counts were recorded at 2 minute intervals, the following parameters were determined: total activity and total mobile counts.

Sacrifice and pathology:

Range-finding phase
All animals were subjected to necropsy. A full macroscopic examination was performed and all lesions were recorded.

Main study
All adult animals including decedents were subjected to necropsy. Adult scheduled necropsies were performed after an overnight period without food. Where possible, the male necropsies were carried out in cage order. The scheduled female necropsies were carried out in ascending group order among the females reaching Day 5 post-partum each day, where possible. A full macroscopic examination was performed and all lesions were recorded. The following tissues were preserved in 10% neutral buffered formalin unless otherwise stated: adrenals, animal identification, aorta, bone marrow smear; femur (fixed in methanol), brain, caecum, colon, duodenum, femur with bone marrow and stifle joint, gross lesions, heart, ileum, jejunum, kidney, larynx, liver, lungs with mainstem bronchi and bronchioles, mandibular lymph nodes, mesenteric lymph nodes, oesophagus, ovaries, oviducts, pancreas, Peyer's patch, pituitary, popliteal lymph nodes, prostate, rectum, sciatic nerves, seminal vesicles, spinal cord cervical, spinal cord lumbar, spinal cord thoracic, spleen, sternum with bone marrow, stomach, testes and epididymides (Bouin's fixative), thymus, thyroids and parathyroids, tongue, trachea, trachea bifurcation, ureters, urinary bladder, uterus including cervix and vagina. All tissues were examined microscopically, for five rats/group from the control and 1000 mg/kg bw/d groups, following embedding in paraffin wax BP, sectioning and staining with haemotoxylin and eosin. Weights were obtained for five rats/group for the adrenals, brain, heart, kidney, liver, mandibular lymph nodes, spleen. Organ weights were obtained for all males for the testes and epididymides.





Bone marrow smears were prepared at necropsy. They were fixed in methanol but not examined.

Other examinations:

Reproductive function was evaluated, this is summarised in Section 7.8.

Statistics:

Body weight gains, necropsy body weights, food consumption, haematology, clinical chemistry, urine analysis, locomotor activity and functional observational battery variables were analysed using one-way analysis of variance (ANOVA), separately for each sex. Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P≥0.01), pairwise comparisons with control were made using Dunnett's test. A linear contrast was used to determine whether there was a relationship between increasing dose and response. A significant trend (P<0.05) was reported only where none of the pairwise comparisons was significant. Organ weights were analysed using Analysis of Covariance (ANCOVA) and Dunnett's test, for each sex separately, using the necropsy body weight as covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Levene's test for equality of variances across the groups was also performed. Where this showed evidence of heterogeneity (male spleen weights; P<0.01), the organ was analysed using one-way ANOVA on absolute organ weights and organ to necropsy body weight ratios.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs related to adverse taste of test material

Mortality:

mortality observed, treatment-related

Description (incidence):

Clinical signs related to adverse taste of test material

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Reductions in 1000 mg/kg bw/d males and females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/d

Food efficiency:

not examined

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

Description (incidence and severity):

Increase in AST and ALT at 1000 mg/kg bw/d males and females, and 300 mg/kg bw/d males

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

reduced mean volume and increased specific gravity in 1000 mg/kg bw/d males

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

decreased mean heart weight in 1000 mg/kg bw/d males

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

mesenteric lymph node; 1000 mg/kg bw/d males and females

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY
There were no treatment-related mortalities. One female in the 1000 mg/kg bw/d was euthanised on Day 1 post-partum due to the severity of
clinical signs (laboured and noisy respiration, semi-closed eyes and sluggish behaviour). At necropsy, moderate distention of the stomach, jejunum, ileum and duodenum was observed along with a slight redness to the mesenteric lymph nodes. As this was an isolated incident and there were no clinical observations or similar macroscopic observations at necropsy for other animals that received the test material, this finding was considered to be incidental and not related to treatment.

CLINICAL SIGNS
Noisy respiration was observed in males and females given 300 or 1000 mg/kg bw/day on several occasions during the dosing period. No correlates were noted at necropsy or microscopic examination therefore it was concluded that this effect was not a direct clinical effect of the test material. Mouth rubbing, salivation and/or paddling of the forelimbs were noted in animals given TOFA_DimerFA_TETA_PAA from immediately post-dose up to the end of the of the day on occasion. The number of animals affected and the duration of the reactions were dose-related. These findings were considered to be a reaction to the adverse taste of the test material. In control animals, mouth rubbing and salivation immediately after dosing was noted in a few animals on isolated occasions during the study.

BODY WEIGHT AND WEIGHT GAIN
There was a statistically significant reduction in mean body weight gain in 1000 mg/kg bw/d males compared to controls, over the first week of the dosing period. Mean body weight gains remained slightly lower than the control group values throughout the dosing period, with a statistically significant reduction for the overall dosing period. There were however, no clinical signs for the males associated with poor clinical condition such as perinasal or peri anal hair staining and there was no effect on mating libido compared with the controls. Therefore lower body weight gain over the six week study period was considered not to have been adverse. There was no treatment related effect on body weight gain for males in the 100 or 300 mg/kg bw/d groups. At 100 mg/kg bw/d, although there was a statistically significant reduction in body weight gain for the dosing period compared with the control group value, due to the lack of a dose-response this was considered to be incidental and not an effect of treatment.
There were no effects of treatment on mean body weight and body weight gain in females during the pre-pairing period. At 100 and 300 mg/kg bw/d, there was no adverse effect of administration on mean maternal body weight gain during gestation or lactation. Over Days 1 to 4 of lactation for females that received 300 mg/kg bw/d there was a statistically significant reduction compared with the control group value. However due to the lack of a
dose-response, this was considered to be incidental and not an effect of treatment.

FOOD CONSUMPTION
Males in the 1000 mg/kg bw/d group exhibited lower mean food consumption during Week 1 of the pre-pairing period (-16.8%). Thereafter mean food consumption improved and was similar to controls. After the pairing period, mean food consumption was similar to the control group values. Lower mean food consumption for males was transient being apparent in Week 1 only and therefore was considered not to be adverse. There were no effects on food consumption in 100 and 300 mg/kg bw/d males. There were no adverse effects on food consumption for treated females during the pre-pairing period. During the first week of the pre-pairing period, mean consumption in treated groups was lower than the control (-3.7%, -13.6% and -10% in groups given 100, 300 or 1000 mg/kg bw/d, respectively) with no dose-relationship. During the second week of the pre-pairing period, mean food consumption values were generally similar to the controls in the groups given 100 or 1000 mg/kg bw/d, with values at 300 mg/kg bw/d remaining lower than the control (-13.4%). Food consumption in treated groups was similar to that of the controls during gestation and lactation.

HAEMATOLOGY
All changes in males and females (including those reaching statistical significance) were considered not to be of toxicological significance due to the absence of a dose-relationship, inconsistency between the sexes and/or the minor nature of the findings, which were considered to represent normal biological variation.

CLINICAL CHEMISTRY
In males and females given 1000 mg/kg bw/d, there was a statistically significant increase in alanine aminotransferase and an increase in aspartate aminotransferase concentration, which was statistically significant in males. There was a single 1000 mg/kg bw/d female with with very high values that may have been spurious (AST 427 IU/L and ALT 160 IU/L). There were also slight increases in inorganic phosphate, urea and total cholesterol concentration. At 300 mg/kg bw/d, there were slight increases in aspartate aminotransferase and alanine aminotransferase (statistically significant in males) concentrations in males and females, compared with control values. These findings were considered to be related to treatment. All other changes in males and females (including those reaching statistical significance) were not considered to be test article related due to the absence of a dose-relationship, inconsistency between the sexes and/or the minor nature of the findings, which were considered to represent normal biological variation for animals of this strain and age.

URINALYSIS
Males in the 1000 mg/kg bw/d group had statistically significant reduction in mean urine volume and increase in specific gravity, compared with controls. There had been no qualitative observation in the clinical recordings that males on this study were consuming more or less water than the controls and there were no kidney pathology findings, therefore this finding was considered not to be adverse. In males given 100 or 300 mg/kg bw/d, mean urinalysis data was comparable to the control group values.

NEUROBEHAVIOUR
There were no effects of treatment on the functional and neurological tests performed. Group mean locomotor activity data were highly variable. Males in the 1000 mg/kg bw/d group were generally more active than controls, with statistical significance over the first 2 minutes of assessment, and overall (101.3% increase in mobile counts). In the absence of similar findings in females (25% increase in overall mobile count compared with the control) and with no male changes in activity or reflexes observed during FOB assessment, this was considered unlikely to be biologically significant.
Locomotor activity in males and in females receiving 100 or 300 mg/kg bw/d was generally comparable with the control group.

ORGAN WEIGHTS
There was a statistically significant decrease in mean heart weight relative to body weight in the 1000 mg/kg bw/d males, compared to controls. However there were no histopathological changes and no statistically significant changes were observed in females, therefore this finding was considered not to be adverse.

GROSS PATHOLOGY
There were no effects of treatment noted at necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
In the mesenteric lymph node, increased histiocyte foci were present in all male and most female rats treated with 1000 mg/kg bw/d. Histiocyte foci were characterised by discrete aggregates of plump eosinophilic macrophages. There were no other treatment related microscopic findings.

OTHER FINDINGS
Range-finding study results
There were no unscheduled deaths, and no clinical signs of toxicity. Mouth rubbing was noted in the majority of animals in all groups (including control) throughout the dosing period upon the return to cage after dosing. Salivation and/or paddling of the forelimbs were observed on several occasions throughout the dosing period in males and females given TOFA_DimerFA_TETA_PAA. These observations are considered to be a reaction to the taste of the test material and/or vehicle. There was a slight dose-related reduction in male body weight gain at 300 and 1000 mg/kg/day, throughout the dosing period, compared with the control group. In females at 1000 mg/kg/day, there was a slight reduction in mean body weight gain over the first 7 days of dosing. However, overall body weight gain for the dosing period was similar to the control group value. Mean food consumption was lower in 300 and 1000 mg/kg bw/d males compared to controls throughout the dosing period. Mean food consumption was slightly lower in 1000 mg/kg bw/d females compared to controls over the dosing period. There were no treatment related effects on haematology parameters. There was a slight increase in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in treated rats, compared with the control group. For AST there was some overlap in the individual animal values between the control and 1000 mg/kg bw/d animals, but for ALT individual values for animals in the 1000 mg/kg bw/d group were consistently higher than the controls. It was therefore considered that the changes in AST and ALT plasma values were related to treatment with TOFA_DimerFA_TETA_PAA and may be indicative of liver damage. There were no macroscopic findings that were considered to be due to test material administration.

Effect levels

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at highest dose level

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. AST and ALT values for rats treated with 0, 300 or 1000 mg/kg bw/d TOFA_DimerFA_TETA_PAA

Sex (dose level)	Aspartate aminotransferase (AST)		Alanine aminotransferase (ALT)
	Mean IU/L (% increase from control)	Range of individual values IU/L	Mean IU/L (% increase from control)	Range of individual values IU/L
Male (0 mg/kg bw/d)	70	58-81	39	31-48
Male (300 mg/kg bw/d)	79 (12.9%)	59-96	51* (30.7%)	36-65
Male (1000 mg/kg bw/d)	98** (40%)	91-111	102*** (161.5%)	94-109
Female (0 mg/kg bw/d)	70	59-83	37	31-45
Female (300 mg/kg bw/d)	75 (7%)	70-82	50 (35%)	41-60
Female (1000 mg/kg bw/d)	156 (122%)	74-112, 427	104*** (181%)	66-160

* p<0.05, ** p<0.01, *** p<0.001

Table 2. Incidence of histiocyte foci: mesenteric lymph node - terminal kill

		Males				Females
Tissue and finding	Dose level (mg/kg bw/d)	0	100	300	1000	0	100	300	1000
Mesenteric lymph node	No. examined	5	0	0	5	5	0	0	5
histiocyte foci	Grade -	5	0	0	0	5	0	0	1
	Grade 1	0	0	0	2	0	0	0	4
	Grade 2	0	0	0	3	0	0	0	0

Key: Grade - = finding not present, Grade 1 = minimal, Grade 2 = slight

Applicant's summary and conclusion

Conclusions:

No adverse effects were observed at the highest dose level administered, therefore the NOAEL was considered to be 1000 mg/kg bw/d in male and female rats.

Executive summary:

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test was conducted in male and female Crl:WI(Han) rats, according to OECD Test Guideline 422. The test material, TOFA_DimerFA_TETA_PAA, was administered orally by gavage. A range-finding study was conducted initially with groups of 3 rats/sex in which the test material was administered daily by gavage for 14 days at dose levels of 0, 100, 300 and 1000 mg/kg bw/day. Based on the findings of the range-finding study, dose levels of 100, 300 and 1000 mg/kg bw/day were selected for the main study.

In the main study, groups of 10 male rats were dosed once daily for two weeks prior to pairing, during the pairing period and a further two weeks before necropsy. Males were treated for a minimum of 6 weeks prior to necropsy. Groups of 10 female rats were dosed for two weeks prior to pairing, during pairing and until Day 4 post-partum (i.e. approximately 7 weeks of dosing). The females were allowed to litter and rear their offspring to Day 4 post-partum. The following parameters were assessed during the study: clinical observations, body weight, food intake, functional observations and tests, locomotor activity, haematology, clinical chemistry, organ weights, gross pathology and histopathology.

There were no treatment related deaths during the study. One female treated at 1000 mg/kg bw/day was euthanised on Day 1 post-partum, but this death was not considered to be treatment related. While noisy respiration was observed on several occasions during the dosing period in males and females treated at 300 or 1000 mg/kg bw/day, it was considered that this finding was not a direct clinical effect of the test material. Occasionally, mouth rubbing, salivation and/or paddling of the forelimbs were noted in animals from immediately post-dose until the end of the day. The number of animals affected and the duration of the reactions were dose-related. These findings were considered to be a reaction to the taste of the test material and of no toxicological significance.

There was a statistically significant reduction in mean body weight gain over the first week of the dosing period in males treated at 1000 mg/kg bw/day. A lower mean body weight persisted throughout the dosing period and was associated during Week 1 and 2 with a lower mean food consumption (compared to controls). It was considered that these changes did not adversely affect the males when clinical signs, functional observations and mating performance were assessed. For females, mean body weight gain throughout the majority of the study was comparable to controls, and although lower food consumption was observed during the first week of the study there was no dose-relationship. There was a statistically significant reduction in mean body weight gain during the last week of gestation in females treated at 1000 mg/kg bw/day, but in the absence of other effects this transient observation was considered not to be adverse. Group mean locomotor activity data were highly variable. Males in the1000 mg/kg bw/day dose group showed statistically increased activity compared to the controls, but in the absence of other effects observed during FOB assessment, it was considered unlikely that this was of biological significance.

There was a statistically significant increase in alanine aminotransferase levels in males treated at 300 and at 1000 mg/kg bw/day and in females treated at 1000 mg/kg bw/day. There was also an increase in aspartate aminotransferase levels in males and in females treated at 1000 mg/kg bw/day; a finding that may indicate liver changes. There were also slight increases in inorganic phosphate and total cholesterol concentration, achieving a significant dose-response. In the absence of changes in liver weight and microscopic morphological liver changes, these findings were not considered to be adverse. There was a statistically significant reduction in mean urine volume and an increase in specific gravity in males treated at 1000 mg/kg/day, compared with the controls. With only a single measurement and with no visual observations of lower water consumption during the in-life phase, this finding was considered to be incidental.

In males treated at 1000 mg/kg/day, there was a statistically significant decrease in mean heart weight relative to body weight, when compared with the controls. There were no histopathological changes or functional changes (FOBs) and no statistically significant changes in female heart weight. In the mesenteric lymph node, increased histiocyte foci were present in all male and most female rats treated with TOFA_DimerFA_TETA_PAA at 1000 mg/kg/day. Histiocyte foci were characterised by discrete aggregates of plump eosinophilic macrophages. These changes were considered to be not adverse.

Based on these findings, the no-observed-adverse-effect-level (NOAEL) for general and systemic toxicity after approximately 6 weeks of dosing in males and 7 weeks of dosing in females was considered to be 1000 mg/kg bw/day (i.e. the highest dose tested).