Registration Dossier

Toxicological information

Toxicity to reproduction

Currently viewing:

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern proprietary study performed to current guidelines under GLP.
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Terephthalic acid obtained from BP Chemicals Limited.
Physical state; white crystals
CTL test substance reference number; Y00751/004
Purity 100%

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
The test animals used were rats, Alpk:APfSD(Wistar-derived)
- Source: Rodent Breeding Unit, Astrazeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire, UK.
-supplied as weanlings (21-23 days old). The weight range at the start of the dosing was 118-189 g (males) and 103-167 g (females).
-28 litters of 4 males per litter and 28 litters of 4 females per litter.

The rats were housed, sexes separately, in multiple rat racks. They were housed in litters initially, two males or two females per cage after they had been assigned to experimental groups and during the pre-mating period; one male was housed with one female for mating; females were housed individually during gestation and with their litter during lactation and were provided with shredded paper bedding material. (After mating, males were housed up to four per cage). Males and females from the same group were housed in adjacent cages during the period prior to mating, to avoid anoestrus.

The animals were housed in cages with the following conditions;
- Temperature 22°C
- Humidity 30-70%
- Air changes: At least 15 changes/hour
- Artificial light giving 12 hours light, 12 hours dark.

Food and water was supplied by an automatic system and were available ad libitum. Water was supplied by water bottles during late gestation and lactation.

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The rats were fed the experimental diet from the first day of study (F0 parents) and throughout the study for the second (F1) generation parents. Males were fed the experimental diet throughout the pre-mating and mating periods and until termination. Females were fed the experimental diet throughout the pre-mating, mating, gestation and lactation periods.

The duration of the pre-mating period was ten weeks from the start of the study for the F0 parents and at least ten weeks from selection for the second (F1) generation parents.
Details on mating procedure:
In each generation, the females were mated with males of the same group for up to 14 days, but in all pairings, brother-sister mating was avoided.
For mating each male was mated with one female by swapping individuals from same-sex pairs in adjacent cages.

A vaginal smear was performed on the female rats. Any female with a positive vaginal smear was immediately separated from the male and housed indindividually. The day that a positive smear was observed was denoted day 1 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from all dietary levels (including controls) were taken prior to the start of the study and at approximately 2 month intervals throughout the study, and analysed quantitatively for terephthalic acid. Prior to feeding the experimental diets, the homogeneity of terephthalic acid in the CTl diet
was determined by analysing samples from the low and high dose levels. The chemical stability of terephthalic acid in diet was determined for these diets over a period of up to 44 days at room temperature and in the freezer (nominal -20C).
Duration of treatment / exposure:
The rats in each generation were fed treated diets continuously from selection until termination. F0 animals were fed experimental diets from 5 weeks of age to termination. Fl offspring were fed experimental diets from birth until termination.
Frequency of treatment:
The feed was available ad libitum.
Details on study schedule:
The first generation pups (F1) were retained with their dams until day 29 post partum at which time twenty six males and twenty six females per group were selected from all appropriate litters. The genealogy of the pups were recorded and taken into account druing the selection procedure. The pups selected to be the parents of the next generation were weighed on day 29 post partum to give the initial F1 generation parent bodyweight values.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000, 5000, 20000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
26 male rats and 26 female rats per dose level
Control animals:
yes, concurrent no treatment
Details on study design:
The study consisted of one control and three treatment groups each containing 26 male and 26 female rats. Treatment started with the original parental animals and was continuous throughout the study until termination of the F2 generation.
Positive control:
No positive control used; not generally required for this study type

Examinations

Parental animals: Observations and examinations:
All animals were examined prior to the start and on each day of the study for changes in clinical condition and behaviour.
Oestrous cyclicity (parental animals):
No examinations performed
Sperm parameters (parental animals):
Sperm motility, number and morphology were assessed immdediately post mortem in all parental males. Homogenistaion resistant spermatids were assessed in frozen testis samples.
Litter observations:
Litters were examined for dead or moribund pups at least once daily and any such pups were subjected to a gross examination post mortem. A count of all live and dead pups was made within 24 hours of parturition (day 1) and thereafter on days 5, 8, 15, 22 and 29 post partum. The sexes of the pups were also recorded at these times.

Individual pup bodyweights were recorded within 24 hours of birth (day 1) and on days 5, 8, 15, 22 and 29 post partum.

On day of birth, the ano-genital distance was measured and recorded for all F1 and F2 pups.

All male pups were examined on day 13 post partum and the presence or absence of nipples or areolae was recorded.

For selected Fl animals the age at which vaginal opening (checked daily from day 29 post partum) and preputial separation (checked daily from day 39 post partum) occurred were determined.
Postmortem examinations (parental animals):
All males were sacrificed after completion of the mating period while females were terminated on or soon after day 29 post partum. The weights of the following organs were noted; adrenal glands, brain, epididymis, kidneys, liver, ovaries, pituitary, prostrate, seminal vesicles, spleen, left and right testis, thymus and uterus.

Following the death of each male, the sperm motility, number and morphology was investigated.

The number of implantation sites was recorded for all females which had been mated. For any female where implantation sites could not be clearly seen in the uterus, ammonium polysulphide was used to determine pregnancy status.

The follwoing tissues were taken from all animals; abnormal tissue, adrenal glands, brain, cervix, coagulating gland, left epididymis and cauda, kidney, liver, ovary, pituitary, prostate, seminal vesicle, spleen, left testis, thymus, uterus with oviducts, vagina and urinary bladder. Tissues from ten animals per sex from each generation from the control and high dose groups were routinely processed, embedded in paraffin wax, sectioned and stained.

All submitted tissues (except those stored) from ten animals per sex, from each generation, from the control and high dose groups, plus those from suspected infertile animals were examined by light microscopy. A quantitative evaluation of primordial follicles including small growing follicles was conducted in the left ovary for 10 Fl females in the control and high dose groups only.

The reproductive organs examined histologically from animals (not included in and therefore additional to the first ten of each sex from the control and high dose groups) suspected of reduced fertility comprised the following: adrenal glands, cervix, coagulating gland, left epididymis and cauda, right ovary, pituitary gland, prostate gland, seminal vesicle, left testis, uterus with oviducts, vagina
Postmortem examinations (offspring):
For pups killed or found dead up to 18 days of age, a visceral examination was carried out, abnormalities were recorded and the pups were discarded.
The weights of the following organs were recorded from one male and one female pup per litter from all litters surviving to scheduled termination; brain, spleen, thymus and kidneys.

Three males and three female pups per group from each litter were selected randomly for a macroscopic examination post mortem.

Pups killed or found dead which were 18 days of age or over were subjected to a full examination post mortem. Abnormal tissues were taken from thepups, fixed and examined by light microscopy.

Where litter size permitted, the following were taken from 3 males and 3 females per litter in all groups and preserved in saline; abnormal tissues and kidneys. Tissues from one pup per sex and litter were taken of the thymus, brain and spleen.
Statistics:
See details below
Reproductive indices:
Mating success, gestation length, pre-coital interval
Offspring viability indices:
A count of all live and dead pups was made within 24 hours of parturition (day 1) and thereafter on days 5, 8, 15, 22 and 29 post partum.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
predominantly in the 20000 ppm group
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
: lower bodyweights at 20000 ppm and ocassionally at 5000 ppm
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
: lower bodyweights at 20000 ppm and ocassionally at 5000 ppm
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL OBSERVATIONS AND MORTALITY; there were two F0 females which were killed due to parturition difficulties ( one in each of the 1000 and 20000 ppm groups). Clinical observations in F0 and F1 animals were red/brown staining on the tray papers below the cages, which was seen predominantly in males in the 20000 ppm group. Pale faeces were recorded for F0 animals receiving 20000 ppm terephthalic acid.

BODYWEIGHT;
Pre-mating F0: There was no effect on bodyweight in the 1000 ppm group. At 5000 ppm, there was an efffect on the male body weight but not the female bodyweight. In the 20000ppm group, the bodyweights were up to 7.5% lower than that of the control group at week 5. Weights of F0 females in the 20000ppm were slightly lower than those of controls.

F1: Bodyweights of males and females receiving 20000 ppm were consistently lower than weights of control. Weights of F1 males receiving 1000 or 5000 ppm terephthalic acid were similar to controls throughout the study. Weights of females receiving 1000 or 5000 ppm were lower than controls throughout the study.

Gestation: F0 bodyweights were slightly reduced (5% below control) in the 20000 ppm group only on day 22 of gestation. There were no effects on bodyweight during gestation in the 1000 or 5000 ppm groups.

Bodyweights of F1 females receiving 20000 ppm terephthalic acid were 9% lower than controls on day 1 of gestation.The difference between the groups increased to a 12% reduction by day 22 of gestation. There was no effect on gestation weight in the 1000 and 5000 ppm group.

Post partum: F0 bodyweights were slightly reduced in the 20000 ppm group on day 1 post partum. The maximum difference from control was 8% on day 15 post partum. There were no effects on bodyweight post partum in the 1000 or 5000 ppm groups.

Fl bodyweights were reduced (10% below controls) in the 20000ppm group on day 1 post partum. The maximum difference from control in this group was 12% on day 15 post partum. Initial weights of animals receiving 5000 ppm were similar to control but subsequently weights were slightly lower than control. There were no effects on bodyweight post partum in the 1000 group.

FOOD CONSUMPTION
Pre-mating;
F0 Food consumption was not affected by treatment.
F1 - food consumption by males and females of the F1 generation receiving 20000 ppm terephthalic acid in the diet was generally lower than that of controls. Food consumption of the 1000 and 5000 ppm group was similar to control.

Gestation;
F0 females in the 20000 ppm group had slightly lower food consumption than controls during the third week of gestation. Food consumption in the 1000 and 5000 ppm group was similar to control.
F1 females in the 20000 ppm group had slightly lower food consumption than controls during the second and third weeks of gestation. Food consumption was similar to the control.

Post partum; Food consumption was reduced in both generations of the 20000 ppm treatment group during the second week post partum but was similar to control in the 1000ppm and 5000ppm treatment groups.

REPRODUCTIVE PERFORMANCE
Smear cycle and pattern; no differences between control and treated F0 and F1 females.

Pre-coital interval: the majority of rats in all groups and both generations mated within the first 4 days of pairing and there was no evidence of an efffect of terephthalic acid on precoital interval.

Gestation length: the majority of females in all groups and both generations littered 22 days following detection of a sperm positive smear. There was no evidence of an effect of terephthalic acid on this parameter.

Proportion of successful matings; the number of successful matings was between 21 and 25 out of a possible 26 in all groups in both generations. There was no evidence of an effect of terephthalic acid on this parameter.

Whole litter losses; Three F1 litters and one F2 litter were lost (all pups dead or missing presumed to have been cannibalised by the dam). The incidence was unrelated to treatment.

Pups live born: The proportion of pups live born was at least 94% in all groups. There was no evidence of an effect of terephthalic acid on this parameter.

Pup survival and litter size: There was no effect on pup survival and litter size in the groups receiving terephthalic acid.

POST MORTEM EXAMINATION
Differences in the organ weight which have been related to treatment are;
1. kidney weights were decreased in all treated male groups in both generations. Effects in females were less consistent and generally absolute values were statistically significantly reduced but values adjusted for bodyweight were not. There was no effect in F0 females in the 1000 ppm group.
2.Liver weights (adjusted for body weight) were increased in both sexes and both generations in the 20000 ppm group only.

There were further differences in organ weight recorded for the ovaries, prostate, brain, pituitary, spleen (both sexes) and thymus (female) for the F0 and F1 generation but these have been attributed to the differences in body weight. Once the numbers were adjusted for bodyweight there was no difference.

Necropsy

Gross abnormalities were observed in the bladder of a few F0 males and one F0 female receiving 20000 ppm terephthalic acid. The individual findings of multiple masses (1 male), firm deposits (2 males), blood stained or discoloured urine (one male and one female) occurred at low incidences but collectively indicate a possible treatment-related effect on the bladder. In addition, blood stained urine was described in one male receiving 5000 ppm terephthalic acid. A much higher incidence of macroscopic abnormalities was observed in the urinary bladder of Fl animals receiving 20000 ppm terephthalic acid. Thickening of the bladder wall and/or prominent blood vessels were observed in nearly half the males and a smaller number of females. Firm deposits were also observed in 3/26 males at this dose level. A few other macroscopic findings were observed at low incidences in FO and Fl animals receiving 20000 ppm terephthalic acid. A low incidence (5/26) of scabs with or without subcutaneous swelling was observed on the tail of F0 males receiving 20000 ppm terephthalic acid but not in any other treated or control group. This is a common spontaneous finding in laboratory rats and since the occurrence was not repeated in the Fl generation it is considered to be unrelated to treatment with terephthalic acid. There was no effect of treatment on the number of mated F0 or Fl animals failing to produce litters, and the macroscopic observations indicated diverse reasons, unrelated to treatment, for the small number of pairs failing to breed successfully.

There was no effect on the number of implantation sites or on post implantation loss in any of the F0 or Fl dams receiving terephthalic acid

Histopathology

No changes were detected in the reproductive organs of F0 or Fl animals which could be attributed to treatment

A variety of changes were observed in the urinary bladder of animals of both sexes receiving 20000 ppm terephthalic acid. The lesions comprised transitional epithelial hyperplasia, cystitis, inflammatory or mononuclear cell infiltration and haemorrhage. The incidence in the F1 animals was greater than in the F0 generation, possibly reflecting the longer period of exposure or the younger age at which exposure to terephthalic acid commenced. It is considered that these changes are related to treatment and indicate an irritant effect of the compound on the bladder mucosa at this dose level. Bladders were not examined from animals receiving 1000 or 5000 ppm. Minimal or slight renal papillary necrosis was observed in the grossly abnormal kidneys of a few males (two in each generation) receiving 20000 ppm terephthalic acid. This is an uncommon spontaneous finding and it is considered that this lesion is likely to be related to treatment. Only macroscopically abnormal kidneys were examined. No changes were observed in any other tissue which could be attributed to treatment.

The number of primordial follicles in the left ovary was assessed for 10 control and 10 high F1 females. At termination, the numbers of small follicles in the ovary of F1 females receiving 20000 ppm terephthalic acid were comparable to control counts

Sperm analysis

There was a higher percentage of motile sperm in F0 males receiving 1000 ppm terephthalic acid in diet. However there was no similar difference from control in F1 males or in the higher dose group and this is considered to be unrelated to treatment. There were higher number of sperm per g right cauda in F0 males receiving 20000 and 1000 ppm terephthalic acid in the diet. However there was no similar difference from control in F1 males and this is considered to be unrelated to treatment. The number of homogenisation resistant spermatids in the right testes was assessed for 10 control and 10 high dose males in each generation and was unaffected by treatment. There was a higher incidence of abnormally sized tails in F0 males receiving 20000 ppm terephthalic acid in the diet. However, there was no similar difference from control in F1 males and this is considered to be unrelated to treatment.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
20 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no effects seen on reproduction and development. Equivalent to 2010.9 -2324.3 mg/kg bw/d achieved pre-mating dose levels)
Remarks on result:
other: Generation: all (migrated information)
Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on COT evaluation
Remarks on result:
other: Generation: both parental (migrated information)
Key result
Dose descriptor:
NOAEC
Effect level:
5 000 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on COT evaluation
Remarks on result:
other: Generation: both offspring (migrated information)

Results: P1 (second parental generation)

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
no effects observed

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
gross pathology
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
: lower at 20000 ppm from Day 15 post partum and at 5000 ppm from Day 29
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

There was no effect on sex distribution in any treated group. The ano-genital distance in female offspring in both generations was not affected in 10000 and 5000 ppm group, however it was statistically significantly lower in the 20000 ppm treatment group than the control.

There were few clinical observations and none were considered to be related to treatment.

Changes in bodyweight were noted. Weights of F1 pups receiving 20000 ppm terephthalic acid were statistically significantly lower than control from day 15 post partum. At days 5 and 8, F1 pups in the 5000 ppm group did have a slightly higher weight than the control pups but by day 29, bodyweights in males were statistically lower than controls. Bodyweights of F1 pups receiving 1000 ppm were similar to control. Bodyweights of F2 males and females in the 20000 ppm group were lower than control at all timepoints.

Nipple/areolae was not affected in any male pups. However, in the female pups the vaginal opening was slightly delayed by 1.6 days in F1 females in the 20000 ppm group. Preputial separation was delayed in F1 males in the 5000 and 20000 ppm groups.

POST MORTEM
There were no observations which were considered to be related to treatment in post mortems of pups up to 18 days of age.
There was a decrease in kidney weight (adjusted for bdoyweight) in all treated groups in both generations. Absolute spleen weights were lower than control values in both generations in the 20000 ppm treatment group, even after adjustment for bodyweight. However, brain and thymus weights were lower than control in both generations for the treatment group, 20000 ppm after bodyweight adjustment there was no difference.

No gross changes were observed in F1 or F2 pups at termination which could be related to terephthalic acid.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
Remarks on result:
not determinable due to absence of adverse toxic effects

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
sexual maturation
clinical signs
mortality
body weight and weight gain
Remarks on result:
not determinable due to absence of adverse toxic effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Selected organ weights for the F0 parents.

Organ weights (mean±SD)

Dietary concentration of terephthalic acid (ppm)

0 (control)

1000

5000

20000

Males - n

26

26

26

26

  Terminal Bodyweight (g)

562.2±50.0

551.2±39.2

557.3±61.4

518.0±57.3

Females - n

26

25

26

25

  Terminal Bodyweight (g)

338.2±16.8

339.1±25.9

336.9±22.6

318.6±26.8

 

 

 

 

 

Adrenal Glands - Males

  Organ weight (g)

0.062±0.010

0.057±0.009*

0.060±0.009

0.056±0.010**

  Organ to bodyweight ratio (%)

0.011±0.001

0.010±0.002

0.011±0.002

0.011±0.002

  Organ weight adjusted for bodyweight

0.062

0.056*

0.060

0.056*

Adrenal Glands - Females

  Organ weight (g)

0.076±0.013

0.080±0.009

0.075±0.012

0.071±0.014

  Organ to bodyweight ratio (%)

0.022±0.004

0.024±0.004

0.022±0.003

0.022±0.005

  Organ weight adjusted for bodyweight

0.076

0.081

0.075

0.070

Kidneys - Males

  Organ weight (g)

3.64±0.29

3.41±0.25**

3.29±0.28**

3.07±0.38**

  Organ to bodyweight ratio (%)

0.65±0.03

0.62±0.04

0.59±0.04

0.59±0.05

  Organ weight adjusted for bodyweight

3.58

3.39**

3.24**

3.20**

Kidneys - Females

  Organ weight (g)

2.48±0.21

2.48±0.25

2.35±0.18**

2.33±0.25**

  Organ to bodyweight ratio (%)

0.73±0.05

0.73±0.03

0.70±0.04

0.73±0.05

  Organ weight adjusted for bodyweight

2.44

2.43

2.32**

2.45

Liver - Males

  Organ weight (g)

20.2±2.6

19.8±2.1

20.2±2.7

19.2±2.4

  Organ to bodyweight ratio (%)

3.6±0.3

3.6±0.3

3.6±0.3

3.7±0.2

  Organ weight adjusted for bodyweight

19.7

19.7

19.8

20.4*

Liver - Females

  Organ weight (g)

16.0±2.7

16.6±2.9

16.1±2.5

15.4±2.6

  Organ to bodyweight ratio (%)

4.7±0.7

4.9±0.7

4.8±0.5

4.8±0.6

  Organ weight adjusted for bodyweight

15.5

16.0

15.8

16.8*

Spleen - Males

  Organ weight (g)

1.125±0.126

1.047±0.120**

1.069±0.125

0.980±0.105**

  Organ to bodyweight ratio (%)

0.201±0.019

0.190±0.019

0.193±0.021

0.190±0.016

  Organ weight adjusted for bodyweight

1.103

1.041**

1.054*

1.022**

Spleen - Females

  Organ weight (g)

0.780±0.070

0.818±0.136

0.790±0.087

0.708±0.079**

  Organ to bodyweight ratio (%)

0.231±0.018

0.241±0.032

0.235±0.022

0.223±0.025

  Organ weight adjusted for bodyweight

0.769

0.805

0.781

0.743

Ovaries - Females

  Organ weight (g)

0.109±0.021

0.107±0.018

0.104±0.021

0.093±0.025**

  Organ to bodyweight ratio (%)

0.032±0.007

0.032±0.006

0.031±0.006

0.029±0.008

  Organ weight adjusted for bodyweight

0.109

0.107

0.104

0.094*

Prostate Gland - Males

  Organ weight (g)

0.646±0.135

0.662±0.145

0.643±0.131

0.574±0.112*

  Organ to bodyweight ratio (%)

0.116±0.029

0.121±0.028

0.117±0.030

0.113±0.026

  Organ weight adjusted for bodyweight

0.647

0.662

0.644

0.572*

** Statistically significant difference from the control group at the 1% level (Student's t-test, two-sided).

* Statistically significant difference from the control group mean at the 5% level (Student's t-test, two-sided).

Comparision of Pups Live Born, Including Whole Litter Losses

 

Dietary concentration of terephthalic acid (ppm)

0 (control)

1000

5000

20000

F1 Litter - n

24

24

24

21

  Prop. Of pups born live

265/270

281/298*

268/275

235/237

  Percentage (mean±SD)

98.4±3.2

94.4±20.3

97.5±5.7

99.3±2.2

  Prop. Of litters with all pups born live

119/24

18/24

19/24

19/21

F2 Litter - n

22

25

23

23

  Prop. Of pups born live

256/267

306/317

256/260

295/295**

  Percentage (mean±SD)

95.9±9.0

96.9±5.9

95.0±20.8

100.0±0.0

  Prop. Of litters with all pups born live

16/22

17/25

20/23

23/23**

** Statistically significant difference from the control group at the 1% level (Student's t-test, two-sided).

* Statistically significant difference from the control group mean at the 5% level (Student's t-test, two-sided).

Applicant's summary and conclusion

Conclusions:
Dietary administration of 20000 ppm terephthalic acid for two successive generations did not result in any effects on reproductive performance. No gross or microscopic changes were seen in the reproductive system that could be related to terephthalic acid administration. Irritant changes were observed in the bladder of males and females receiving 20000 ppm terephthalic acid and there was some evidence for an effect on the kidney at this dose level. These tissues were not examined for the 1000 or 5000 ppm groups. Reductions in pup bodyweight generally occurred from day 15 post partum, when the offspring had started consuming solid diet, and are considered to be a direct effect of the test material on the pups rather than an expression of developmental toxicity. Pup bodyweights in the F2 generation of the 20000 ppm treatment group were lower than control from parturition,
but this is considered to be related to the larger litter size in this group. The only effect at a dose level of 1000 ppm was a decrease in kidney weight in adults and pups. The no observed adverse effect level (NOAEL) for effects on reproduction and development was 20000 ppm, the highest dose used in this study.
Executive summary:

The potential of terephthalic acid to cause reproductiove toxicity was investigated in a modern, guideline and GLP-compliant two generation study.

Groups of 26 male and 26 female (F0) parents) weanling Alpk:APfSD (Wistar-derived) rats were fed diet containing 0 (control), 1000, 5000 or 20000 ppm terephthalic acid. After 10 weeks, the animals were mated and allowed to rear the ensuing Fl litters to weaning. The breeding programme was repeated with the Fl parents selected from the Fl pups to produce the F2 litters after a 10-week pre-mating period. Test diets were fed continuously throughout the study. The growth of the parental generation, reproductive function, mating behaviour, conception, gestation, parturition, lactation and weaning and the growth and development of the pup were determined.

Bodyweights were reduced in F0 males, F0 females receiving 20000 ppm terephthalic acid (during gestation and post partum) and in Fl males and females. Weights of animals receiving 1000 or 5000 ppm terephthalic acid were similar to controls throughout the study. Food consumption of Fl males and females receiving 20000 ppm was generally lower than that of controls. Food utilisation was less efficient than control in the 20000 ppm group in both generations. There were no effects on smear cycle and pattern, pre-coital interval, gestation length, proportion of successful matings, pups live born, litter size, pup survival, pup sex distribution or pup clinical observations in any treated group. Bodyweights of Fl pups receiving 20000 ppm terephthalic acid were reduced from day 15 post partum. Bodyweights of F2 males and females in the 20000 ppm treatment group were lower than control at all timepoints which correlated with increased litter sizes compared to control. Total litter weight was not different across all groups. Bodyweights of Fl males receiving 5000 ppm were lower than controls on day 29 post partum. Bodyweights of F2 pups receiving 5000 ppm were lower than control from day 15 post partum. There was no effect on pup weight in the 1000 ppm group.

There was a statistically significant decrease in the ano-genital distance of females only in the Fl and F2 litters in the 20000 ppm dose group. Anogenital distances in the 1000 and 5000 ppm females and in all male groups were similar to those of controls. Vaginal opening was slightly delayed, by 1.6 days, in Fl females in the 20000ppm group. Preputial separation was delayed in Fl males in the 5000 and 20000 ppm groups (by 0.8 days and 1.6 days respectively). These differences were related to the reduced bodyweight of these animals. Kidney weights (both absolute and adjusted for bodyweight) were decreased in males from all treated male groups in both generations. Effects in females were less consistent and generally only absolute kidney weight were reduced. Relative liver weights were increased in both sexes and both generations in the 20000 ppm group only. There were no effects on sperm number, sperm motility or sperm morphology. The number of decedent F0 and Fl animals was very low and the incidence was unrelated to dose level. There was no effect of treatment on the number of mated F0 or Fl animals failing to produce litters and no changes were detected in the reproductive organs which could be attributed to treatment. A variety of changes were observed in the urinary bladder of animals of both sexes receiving 20000 ppm terephthalic acid. The incidence in the Fl animals was greater than in the F0. It is considered that these changes are related to treatment and indicate an irritant effect of the compound on the bladder mucosa. Bladders were not examined from animals receiving 1000 or 5000 ppm. Minimal or slight renal papillary necrosis was observed in the grossly abnormal kidneys of a few males (two F0 and two Fl) receiving 20000 ppm terephthalic acid. This is an uncommon spontaneous finding and it is considered that this lesion is likely to be related to treatment. Only macroscopically abnormal kidneys were examined.

In conclusion, the dietary administration of 20000 ppm terephthalic acid for two successive generations did not result in any effects on reproductive performance. No gross or microscopic changes were seen in the reproductive system that could be related to terephthalic acid administration. Irritant changes were observed in the bladder of males and females receiving 20000 ppm terephthalic acid and there was some evidence for an effect on the kidney at this dose level. These tissues were not examined for the 1000 or 5000 ppm groups. Reductions in pup bodyweight generally occurred from Day 15 post partum, when the offspring had started consuming solid diet, and are considered to be a direct effect of the test material on the pups rather than an expression of developmental toxicity. Pup bodyweights in the F2 generation of the 20000 ppm treatment group were lower than control from parturition, but this is considered to be related to the larger litter size in this group. The only effect at a dose level of 1000 ppm was a decrease in kidney weight in adults and pups. The no observed adverse effect level (NOAEL) for effects on reproduction and development was 20000 ppm, the highest dose used in this study.

COT evaluation

The apparent effects on kidney weight seen in this study were considered by the independent UK Committee on Toxicity (COT). The COT used the criteria for distinguishing adverse from adaptive effects outlined in ECETOC Technical report No 85 (Recognition of, and Differentiation between, Adverse and Non-adverse Effects in Toxicology Studies). COT Members were satisfied that the histopathological data indicated a clear no observed adverse effect level (NOAEL) for histopathological changes in the urinary bladder and kidney (renal papillary necrosis) corresponding to administration of 5000 ppm TPA in the diet in this study. Statistically significantly decreased renal weights (adjusted for bodyweight) were present in all generations including the parental generation. However, given that there was no associated histopathology or effect on renal function, it was not clear whether this effect should be considered adverse. It was also noted that this effect was not observed in a chronic toxicity study using a different rat strain

It was concluded that the effects on renal weight seen at 1000 ppm were without histopathological correlates and were concluded to be adpative effects. The COT therefore defined the NOAEL for toxicity as 5000 ppm (approximately 500 mg/kg bw/d).