Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

2-Generation study (Milburn 2003): NOAEL = 491.6-577.4 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
various studies reported but no specific dates presented in study reported in 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Modern proprietary study performed to current guidelines under GLP.
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes (incl. certificate)
Limit test:
no
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.
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 dietwas 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 during 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.
Remarks:
Doses / Concentrations:0, 1000, 5000, 20000 ppmBasis: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
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 immediately post mortem in all parental males. Homogenisation 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.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
predominantly in the 20000 ppm group
Mortality:
no mortality observed
Description (incidence):
Two F0 females which were killed due to parturition difficulties (one in each of the 1000 and 20000 ppm groups).
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 body weights at 20000 ppm and occasionally at 5000 ppm
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
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. 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.
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
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.

Body Weight: Pre-mating F0; there was no effect on bodyweight in the 1000 ppm group. At 5000 ppm, there was an effect 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 20000 ppm 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. F1 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 effect 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 mating; the number of successful mating 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; 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. 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 F1 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 F0 and F1 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 F1 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 F1 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 F1 dams receiving terephthalic acid.
Histopathology: No changes were detected in the reproductive organs of F0 or F1 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.
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)
Dose descriptor:
NOAEL
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)
Dose descriptor:
NOAEL
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)
Key result
Dose descriptor:
NOAEL
Effect level:
2 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Remarks on result:
other: Absolute spleen weights were lower than control values in both generations in the 20000 ppm treatment group, even after adjustment for bodyweight.
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Changes in bodyweight were noted i.e. 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.
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
There was a decrease in kidney weight (adjusted for bodyweight) 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.
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
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 i.e. 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 bodyweight) 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.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
20 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Reproductive effects observed:
not specified

Intakes of terephthalic acid

 

Mean dose received (mg/kg bw/d)

Males

Females

1000 ppm

5000 ppm

20000 ppm

1000 ppm

5000 ppm

20000 ppm

F0 parents pre-mating

97.6

491.6

2010.9

104.4

520.4

2095.5

F1 parents pre-mating

107.6

536.2

2231.5

112.8

577.4

2324.6

F0 females during gestation

-

-

-

86.3

425.1

1684.9

F1 females during gestation

-

-

-

87.4

430.9

1743.7

F0 females during lactation

-

-

-

241.6

1151.9

4543.4

F1 females during lactation

-

-

-

260.7

1217.6

5072.5

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

  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

Litters with all pups born live

19/24

18/24

19/24

19/21

F2 Litter - n

22

25

23

23

  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

  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).

Conclusions:
Dietary administration of 20000 ppm terephthalic acid (equivalent to 2010.9-2324.3 mg/kg bw/d based on premating consumption) 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 reproductive 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 (equivalent to 2010.9 -2324.3 mg/kg bw/d based on pre-mating consumption) 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 (equivalent to 491.6 -577.4 mg/kg bw/d based on pre-mating consumption) 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 adaptive effects. The COT therefore defined the NOAEL for toxicity as 5000 ppm (approximately 500 mg/kg bw/d).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
491.6 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Modern, guideline-compliant multigeneration study, with supporting data
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

TPA: screening study

In a one-generation screening study (Ledoux et al, 1982), administration of terephthalic acid in the diet at concentrations of up to 5% did not affect reproduction or fertility parameters in CD or Wistar rats. TPA exposure decreased Day 1 bodyweight and 21-day survival of offspring at the highest dietary concentration of 5%.  The incidence of renal and bladder calculi at Day 51 in pups fed the same test diets as their mothers was increased at 2% and 5% TPA.

 

TPA: multi-generation study

In a modern, GLP- and guideline-compliant proprietary study (Milburn, 2003), dietary administration of 20000 ppm terephthalic acid for two successive generations of Wistar-derived rats 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 controls from birth, but this finding is considered to be related to the larger litter size in this group.  The only effect seen at a dose level of 1000 ppm was a decrease in kidney weight in adults and pups.  The NOAEL for effects on reproduction is therefore 20000 ppm (equivalent to 2010.9-2324.3 mg/kg bw/d achieved pre-mating dose levels), the highest dose level used in this study.

 

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

Published study reporting effects on testicular function 

In a non-standard published study, Cui et al (2004) administered terephthalic acid for 90 days to determine the effects on testicular function in male Sprague-Dawley rats.  Terephthalic acid was mixed with a standard rodent diet at levels of 0, 0.2, 1 and 5%.  Testicular function was assessed by histopathology, testicular sperm head counts, daily sperm production, sperm motility, biochemical indices (marker testicular enzymes) and serum testosterone levels.  There were no effects of administration on body weight gain or food consumption.  Damage of spermatogenic cells and Sertoli cells was observed by electron microscopy; testicular sperm head counts, daily sperm production, and sorbitol dehydrogenase (SDH) activity were decreased significantly in the 5% group.  Sperm motility was significantly reduced in all treated groups, in a dose-dependent manner. The authors conclude that the 90-day daily administration of terephthalic acid at levels of up to 5% in the diet can cause impairment of testicular function in male rats.

The findings of the study of Cui et al (2004) are not considered to be of clear toxicological significance given the clear absence of functional reproductive effects in the one-generation screening study at dietary concentrations of up to 5% and in the two-generation study at dietary concentrations of up to 2000 ppm; and also in the clear absence of effects on sperm parameters in the multi-generation study. Based on findings in the screening study and the 90-day toxicity studies, the highest dose level of 5% in the Cui et al (2004) study would be expected to result in toxicity (bodyweight effects), although no signs of toxicity are noted in this paper.  In contrast, signs of toxicity were apparent in the study of Milburn et al (2003) study. The reported effects on sperm at the lower dose levels in the study of Cui et al are not considered to be relevant given the clear absence of effects at dose levels of up to 5% in the screening study and up to 20000 ppm (2%) in the Milburn study.

Read-across study with DEHT/DOTP

Faber et al (2002) report a 2 -generation study performed with the read-across substance DETP / DOHT. No effects on fertility or reproductive capacity were reported at dietary concentrations of up to 1.0% DEHT (10000 ppm, equivalent to a mean achieved intake of 1200 mg/kg bw/d using a default conversion factor). Takin into account the conversion of DEHT to TPA by hydrolyis, the achieved mean intake at the highest dietary concentration of DEHT is equivalent to approximately 510 mg/kg bw/d. The absence of findings in this study is consistent with reliable reproductive toxicity studies with TPA and supports the read-across approach used for other endpoints.


Short description of key information:
No evidence of reproductive toxicity was seen in a modern two-generation study performed with terephthalic acid.

Justification for selection of Effect on fertility via oral route:
Modern proprietary study performed to current guidelines under GLP.

Effects on developmental toxicity

Description of key information
No evidence of developmental toxicity was seen in an inhalation study in rats exposed to TPA at levels of up to 10 mg/m3 (Ryan et al, 1990).
Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January - April 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Please refer to Section 13.2 for full read-across justification
Qualifier:
according to
Guideline:
EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Qualifier:
according to
Guideline:
other: ICH Guideline Section 4.1.3
Deviations:
yes
Remarks:
: one deviation was noted but it did not impact the quality or integrity of the study.
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Details on test animals and environmental conditions:
Test animals:-Source: Charles River Laboratories, Portage, Michigan-Sex: female-Condition at receipt: sexually mature female virgins-Age at receipt: approximately 80 days old-Age at study initiation: approximately 12 weeks-Acclimation period: approximately 6 days -Housing: Animals after the first 6 days of acclimation were single housed in stainless-steel cages. Cage paper was changed at least three times a week during the study.-Diet: PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, with or without test material, ad libitum-Water: Reverse osmosis-purified drinking water was supplied via an automatic watering system, ad libitum-Method of animal identification: Each mouse was uniquely identified by a tail tattooEnvironmental Conditions:-Temperature: 70.4 to 70.6 °F (21.3 - 21.5 °C)-Humidity: 39.7 - 46% relative humidity-Photoperiod: 12 hours light/12 hours dark-Air Exchanges: approximately 10 per hour
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
The control and test diets (1000, 3000 and 7000 ppm) were offered ad libitum and supplied weekly from gestation Days 0-18 to randomly selected groups of 25 pregnant females. Study Schedule:-Experimental start date (animal receipt): January 25, 2005-Experimental start date: February 4, 2005-Test diet administration: February 4-25, 2005-Last laparohysterectomy: February 25, 2005-Experimental termination/completion date: April 18, 2005.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Animals were fed PMI Nutrition International, LLC, Certified Rodent LabDiet 5002, with or without test substance. For test diets, an appropriate amount of test substance and diet for each group was weighed, placed into a Hobart mixing bowl, and mixed for 5 minutes. The premix was transferred to a V-blender and mixed for 10 minutes with enough basal diet to form batches of diets containing 1000, 3000 and 7000 ppm. Dose levels were selected based on a previous range-finding study in which doses of 500 to 10000 ppm were well tolerated in pregnant mice.The test diets were prepared biweekly, placed in labeled high density polyethylene (HDPE) plastic drums with no liners, and stored at room temperature. Diets thus prepared were stable for 15 days, as shown by previous analyses with the test substance. The control diet was weighed biweekly into a plastic bag and placed in a HDPE plastic storage drum with no liner. Prior to use, samples (approximately 100 g each) of each diet (including the control) were taken from the top, middle and bottom strata for homogeneity/ concentration analyses. A GC/FID area % purity determination was also performed. Samples of at least 10 g were collected prior to and after treatment and were analyzed for structure of the test substance. All prepared diets were homogeneous (samples from all strata were within 100-102% of target concentrations) and contained the amount of test substance desired (ranged from 96-106% of target concentrations of 1000, 3000 and 7000 ppm). The substance used in the study was at least 97.6% pure at the end of the study (compared to a purity of 99.79% on the Certificate of Analysis). A GC-MS analysis showed the substance to be authentic di (2-ethylhexyl) terephthalate. No contaminants were present in the feed or water at concentrations sufficient to affect the outcome of the study.
Details on mating procedure:
Females in good health that weighed at least 20 g were paired for mating in the home cage of a resident, sexually mature Crl:CD1(ICR) male from the same source. Each mating pair was examined daily. Following positive evidence of mating (vaginal copulatory plug), the females were returned to their individual cages. The day on which evidence of mating was identified was designated as Gestation Day 0. Actual body weight values ranged from 25.8 to 34.2 g on Gestation Day 0.
Duration of treatment / exposure:
Gestation Days 0-18
Frequency of treatment:
Continous / daily (dietary administration)
Duration of test:
18 days
Dose / conc.:
7 000 ppm
Dose / conc.:
3 000 ppm
Dose / conc.:
1 000 ppm
Dose / conc.:
0 ppm
No. of animals per sex per dose:
25 mated females
Control animals:
yes, plain diet
Details on study design:
-Clinical observations: All mice were observed twice daily for moribundity and mortality. Individual, detailed clinical observations were recorded daily from Gestation Days 0-18. All significant findings were recorded.-Body Weight: All animals were weighed daily from Gestation Days 0-18-Food Consumption: All animals were weighed daily from Gestation Days 0-18-Mean Compound Comsumption: Mean compound consumption (in mg/kg/day) for each group was determined by dividing the concentration of test material in the diet (in mg/kg) by the g/kg/day food consumption value for each interval.
Maternal examinations:
-Clinical observations: All mice were observed twice daily for moribundity and mortality. Individual, detailed clinical observations were recorded daily from Gestation Days 0-18. All significant findings were recorded.-Body Weight: All animals were weighed daily from Gestation Days 0-18-Food Consumption: All animals were weighed daily from Gestation Days 0-18-Mean Compound Comsumption: Mean compound consumption (in mg/kg/day) for each group was determined by dividing the concentration of test material in the diet (in mg/kg) by the g/kg/day food consumption value for each interval.
Ovaries and uterine content:
All surviving mice were euthanized on Gestation Day 18 by CO2 inhalation. The thoracic, abdominal and pelvic cavities were opened and examined, and any abnormalities were recorded. Liver weights were recorded. The uterus and ovaries were exposed and excised. The numbers of corpora lutea on each ovary were recorded. All implantation sites (including resorptions) were numbered in consecutive order (beginning with the left distal to the left proximal uterine horn), noting the position of the cervix, and continuing from the right proximal to the right distal uterine horn. Maternal tissues exhibiting gross changes were retained in 10% neutral-buffered formalin for possible future examination. Uteri with no evidence of implantation were placed in 10% ammonium sulfide solution for detection of early implantation loss.One female in the high dose group delivered on Gestation Day 18. This animal was euthanized and necropsied. The number and location of implantation sites, corpora lutea and viable pups were recorded and included in the mean fetal data. Viable pups from this female were euthanized and examined viscerally and skeletally. A control female was euthanized on Gestation Day 6 in extremis. The uterus had no macroscopic evidence of implantation and was placed in 10% ammonium sulfide. Any grossly affected tissues from this animal were retained in 10% neutral-buffered formalin and subsequently examined.
Fetal examinations:
At necropsy each fetus was weighed, sexed, examined macroscopically for any external findings, and euthanized by an intrathoracic injection of sodium pentobarbital (if necessary). The external examination included (but was not limited to) an examination of the eyes, palate and external orifices. The crown-rump length, weight, and sex of nonviable fetuses (not autolyzed) were determined. Crown-rump measurements and degrees of autolysis were recorded for late resorptions (if present). Each viable fetus was examined viscerally (included examination of the heart and major blood vessels). Fetal kidneys were examined and graded for renal papillae development. Heads from approximately one half of the fetuses in each litter were placed in Bouin's fixative and subsequently examined using the Wilson sectioning technique. The heads from the rest of the animals were examined by a mid-coronal slice. All carcasses were eviscerated and fixed in 100% ethyl alcohol. Following fixation, each fetus was macerated in KOH and stained with Alizarin Red S and Alcian Blue. External, visceral and skeletal findings were recorded as developmental variations (changes representing slight deviations from normal that were considered to have no effect on animal health or body conformity) or malformations (anomalies that alter general body conformity, disrupt or interfere with normal body function, or may be incompatible with life).Fetal data were presented according to the numbers of fetuses and litters available for examination in each group, and the number of affected fetuses per litter on a proportional basis.
Statistics:
Analyses were conducted using two-tailed tests (except where noted) at significance levels of p < 0.05 and 0.01, comparing each test group to the control group. Data were presented as mean ± SD. Mean maternal body weight (absolute and net), body weight changes (absolute and net) and food consumption, gravid uterine weights, numbers of corpora lutea, implantation sites and viable fetuses, fetal body weights (separately and by sex combined) and liver weights were analyzed using a one-way analysis of variance (ANOVA). If the ANOVA revealed a significant intergroup variance, Dunnett's test was used to compare test groups to the control group. Mean litter proportions of prenatal data (viable and nonviable fetuses, early and late resorptions, total resorptions, pre- and post implantation loss and fetal sex distribution) and total malformations and developmental variations (external, visceral, skeletal and combined) and of each malformation or variation were analyzed using the Kruskal- Wallis nonparametric ANOVA test to determine intergroup variances. If the variance was significant, the Mann- Whitney U-test was used to compare test to control data.
Indices:
Intrauterine data were summarized using two methods of calculation. An example of each method of calculation follows:1. Group Mean Litter Basis:No. Dead Fetuses, Postimplantation Loss/Litter = [Resorptions (Early/Late)/Group] / [No. Gravid Females/Group]2. Proportional Litter Basis:Summation Per Group (%) = [∑ Postimplantation Loss/Litter (%)] / [No. Litters/Group]Where:Postimplantation Loss/Litter (%) = [No. Dead Fetuses, Resorptions (Early/Late)/Litter] / [No. Implantation Sites/Litter] x 100The fetal developmental findings were summarized by: 1) presenting the incidence of a given finding both as the number of fetuses and the number of litters available for examination in the group; and2) considering the litter as the basic unit for comparison and calculating the number of affected fetuses in a litter on a proportional basis as follows:Summation per Group (%) = [∑ Viable Fetuses Affected/Litter (%)] / [No. Litters/Group]Where:Viable Fetuses Affected/Litter (%) = [No. Viable Fetuses Affected/Litter] / [No. Viable Fetuses/Litter] x 100
Historical control data:
Historical Control data from the testing laboratory was provided as part of the study report consisting of 22 developmental toxicity/teratogenicity studies.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Reduced body weight gain during Gestation Days 12-13 in the 3000 ppm group (2.0 ± 0.63 g in treated vs. 2.5 ± 0.43 g in control), which was not seen at 7000 ppm.
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Mean absolute liver weights of animals in the 3000 (3.0053 ± 0.35957) and 7000 (3.2516 ± 0.29470) ppm groups were 8.4% (p < 0.05) and 17.3% (p < 0.01) higher than control.
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
Higher mean litter proportion of pre-implantation loss in the 3000 ppm group (6.7 ± 8.18% in treated vs. 3.0 ± 9.21% in control). Since this was not observed in the 7000 ppm group (preimplantation loss was 2.7 ± 5.87%), it was not considered to be related to test material.
Details on maternal toxic effects:
Maternal toxic effects: yes. Details on maternal toxic effects: maternal data with dose level, clinical observations and survival; All animals survived to study termination, with the exception of one high dose animal that was euthanized after delivery on Gestation Day 18 and one control animal that was euthanized in extremis on Gestation Day 6. This animal had facial lacerations and was hypoactive, cool to the touch, and exhibited tremors. At necropsy, this animal was found to have a fractured nasal bone and was nongravid. Clinical findings were limited to single animals and/or did not occur in a dose-related manner. Body weight: Maternal body weight, body weight gain, net body weight, net body weight gains and gravid uterine weights were not affected by treatment with any dose. The only significant change was reduced body weight gain during Gestation Days 12-13 in the 3000 ppm group (2.0 ± 0.63 g in treated vs. 2.5 ± 0.43 g in control), which was not seen at 7000 ppm. Food consumption: There was no effect of test material on food consumption. Gross pathology, incidence and severity: There were no internal findings in the high dose animal that delivered on Gestation Day 18. All females were internally normal, with the exception of one female in the high dose group that had a white area on the liver. The numbers of females that were nongravid in the control, 1000, 3000 and 7000 ppm groups were 3, 4, 4 and 1, respectively. Organ weight changes: Mean absolute liver weights of animals in the 3000 (3.0053 ± 0.35957) and 7000 (3.2516 ± 0.29470) ppm groups were 8.4% (p < 0.05) and 17.3% (p < 0.01) higher than control. Reproductive parameters: The numbers of nongravid females in the control, 1000, 3000 and 7000 ppm groups were 3/25, 4/25, 4/25 and 1/25, respectively (no significant difference). Pre-implantation loss, number of implantation sites and numbers of corpora lutea were not affected by any dose of test material. The only significant change was a higher mean litter proportion of preimplantation loss in the 3000 ppm group (6.7 ± 8.18% in treated vs. 3.0 ± 9.21% in control). Since this was not observed in the 7000 ppm group (preimplantation loss was 2.7 ± 5.87%), it was not considered to be related to test material.
Dose descriptor:
NOAEL
Effect level:
1 000 ppm
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Fetal body weight changes:
no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
External malformations:
no effects observed
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects.
Details on embryotoxic / teratogenic effects: Litter size, weight and sex ratio: There was no effect of treatment on live litter size, fetal body weight or sex ratios. Total numbers of malformations/variations: The numbers of fetuses (litters) available for examination were 265(21), 276(21), 246(21) and 308(24) in the control, 1000, 3000 and 7000 ppm groups, respectively. Malformations (all of which were considered to be spontaneous) were observed in 4(3), 1(1), 8(4) and 3(3) fetuses (litters) in the respective groups. There was no effect of test material on the fetal incidence or mean litter percent with malformations. Grossly visible abnormalities: No external developmental variations were reported in any group. External malformations occurred in the control and 3000 ppm groups only. One fetus in the control group had a meningocele. Four fetuses from one litter in the 3000 ppm group had tarsal flexure and cleft palate, and one fetus each in two different litters from this group had tarsal flexure or cleft palate. Although the litter proportions of these malformations in this group (2.0% and 2.2%, respectively) were higher than study (0% for both malformations) and historical controls (0.6% and 0.7%, respectively), they were not observed in the 7000 ppm group and were primarily clustered in one litter. Therefore, they were not considered to be related to treatment. Visceral malformations and variations: The only visceral malformation noted was an absent kidney and ureter in one fetus in the 3000 ppm group. Visceral variations were noted in single fetuses in the 3000 (retroesophageal right subclavian artery; no brachiocephalic trunk) and 7000 ppm (hemorrhagic iris) groups. None of these changes were considered to be related to administration of test material. Skeletal malformations and variations: Skeletal malformations were noted in three controls (fused sternebrae in two animals and severely malaligned sternebrae in another), one low dose (fused and/or malpositioned costal cartilage), one mid dose (severely malaligned sternebrae) and three high dose fetuses (severely malaligned sternebrae, fused ribs and fused and/or malpositioned costal cartilage). There were no differences in the incidences of these malformations in treated animals vs. controls when evaluated on a percent litter basis. Variations were noted in all groups (including controls) and consisted of 14th rudimentary rib(s), 14th full rib(s), 7th sternebra, accessory skull bones, malaligned sternebrae (slight or moderate), and 7th cervical rib(s). Vertebral centra unossified, extra site of ossification anterior to sternebra No. 1, extra site of ossification anterior to cervical arch No. 2, 25 presacral vertebrae and 27 presacral vertebrae were found in one, two or three fetuses in the 1000, 3000 or 7000 ppm groups. There were no significant differences in the incidences of any skeletal variations between treated and control animals when evaluated by incidence or on a percent litter basis and there was no dose-response relationship.
Dose descriptor:
NOAEL
Effect level:
7 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
skeletal malformations
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
No evidence of developmental toxicity was seen in this study at matrenally toxic dose levels.
Executive summary:

In this study, groups of 25 pregnant CD-1 mice were exposed to di(2-ethylhexyl)terephthalate at dietary concentrations of 0, 1000 , 3000 or 7000 ppm on Gestation Days 0-18. Dietary concentrations were equivalent to mean achieved intakes of 0, 197, 592 and 1382 mg/kg bw/d, respectively. Dams were sacrificed at Gestation Day 18; fetuses were individually weighed and examined for external, skeletal and visceral malformations and variations. The two highest dietary concentrations were maternally toxic, as manifested by higher mean absolute liver weights. There was no evidence of teratogenicity or fetotoxicity at any dietary concentration. Under the conditions of this study, the NOAEL for maternal toxicity was 1000 ppm (197 mg/kg bw/d) and the NOAEL for developmental toxicity was 7000 ppm (1382 mg/kg bw/d).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
January to February, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Published proprietary GLP study, similar to OECD guidelines.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
EPA (40 CFR 792)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
The animals were male and female Sprague-Dawley rats, obtained from Charles River Breeding Laboratories (MI). Males were used for breeding purposed and were not exposed to PTA.Female rats weighed 117-150 g and were group housed (3-4/cage) on receipt, whilst males were singly housed. Mated females were singly housed in suspended polycarbonate cages throughout the study, except during exposure when they were transferred to stainless steel wire mesh inhalation cages. San-I-Cel corncob bedding was used in the polycarbonate cages. Individuals were identified by numbers written onto the base of the tail using non-toxic permanent marker pen and cage cards.The air conditioned animal rooms were maintained at approximately 22°C and 40% relative humidity. Fluorescent lighting was provided on a 12 hour light/dark cycle. Purina Rodent Chow 5001 and reverse osmosis purified water were provided ad libitum (except during inhalation exposures).
Route of administration:
inhalation: aerosol
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test material was ground to respirable sized particles using a Retsch Ultra-Centrifugal Mill (Type ZM1) and was stored in foil-covered glass jars prior to use. The rats were exposed to the test material as a particulate aerosol in air. Control dams were exposed to filtered air only.Target concentrations were 0.0, 1.0, 5.0 and 10.0 mg/m³. The test article aerosol was generated using a dry materials feeder. Test material was placed into the feeder reservoir and moved to the bottom of the feeder by slow peristaltic action of the flexible, tapered walls of the feeder. At the bottom of the feeder, the test article was taken up by a rotating helix and carried to the generator exhaust, where it was blown into the mixing chamber by a Transvector Jet. Three other Transvector Jets drew the generated aerosol from the mixing chamber through tygon lines and blew the aerosol into the tubing leading into the appropriate exposure chambers. The aerosol entered through the top of the exposure chambers, via and venturi tube, and was exhausted through a pipe located near the bottom of the chamber. The air exhausted from the chamber was drawn through a filtering system before being discharged to the outside environment. Exposures were conducted in 2 m³ stainless steel and glass chambers (Hazleton Systems). The chamber air was filtered through HEPA filters and controlled for temperature and humidity. Chamber airflow in the 2 m³ was maintained at 305 to 420 L/min.Chamber temperature, humidity and airflow were measured hourly during each exposure period.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test material concentrations were determined by spectophotometric analysis. The exposure chamber was sampled at least twice during each exposure period. Samples were collected by drawing a known volume of test atmosphere across an open-faced glass fibre filter. The filters were extracted in 6% ammonium hydroxide in reverse-osmosis purified water and the optical density measured. The absorbance of the filter extracts was compared to a standard calibration curve. The particle size distribution of the aerosol in the exposure chamber was determined weekly using an Anderson Cascade Impactor.
Details on mating procedure:
During the mating period, two females and one male were housed together. Matings were confirmed via a sperm-positive vaginal smear. To facilitate teratological evaluation, mating was staggered over seven consecutive weekdays.
Duration of treatment / exposure:
6 hours/day for 10 days (Gestation days 6 to 15)
Frequency of treatment:
Daily
Duration of test:
From mating until Gestation day 20.
Dose / conc.:
10 other: mg/m³
Dose / conc.:
5 other: mg/m³
Dose / conc.:
1 other: mg/m³
Dose / conc.:
0 other: mg/m³
No. of animals per sex per dose:
The study was conducted using four groups of rats with 26-27 timed-pregnant primiparous dams per group.
Control animals:
yes, sham-exposed
Details on study design:
Females determined as having mated, via a sperm-positive vaginal smear, were weighed on gestation day 5 (day 0 being the day the smear was positive) and their weight was used for randomisation. In order to attain a minimum group size of 20 dams, all female rats which mated were randomly assigned to study groups.
Maternal examinations:
Following treatment initiation, dams were observed twice daily on weekdays and once daily on weekends for untoward effects of exposure. Physical examinations were performed prior to study initiation and again immediately prior to study termination. Rats were observed for signs of toxicity approximately 0-3 hours following each exposure on gestation Days 6-15, and daily thereafter. Individual body weights were determined on gestation Days 0, 5, 6, 11, 16 and 20.
Ovaries and uterine content:
All dams were subjected to a Caesarean section and gross necropsy on gestation Day 20. Their pregnancy status was determined along with the presence of lesions and/or abnormal conditions in the dam of foetus. The uterine horns, foetuses and ovaries were removed intact, trimmed free of adherent tissue and weighed. The uterine horns of dams with no observable implants were stained in a 10% ammonium sulphide solution to determine pregnancy/resorption status.
Fetal examinations:
Foetuses were exised from the uterine horns, given identification numbers, and individually weighed after the total number and disposition of each implant was recorded. Each foetus received a gross external morphologic examination. One-half of each litter was randomly assigned to receive either a skeletal or a wet visceral examination by the method of Staples. Foetuses assigned to wet visceral examination were decapitated and the heads were fixed in Bouin's solution and evaluated for cephalic anomalies by the method of Wilson. Each of the skeletal anomalies was evaluated and classified as either a variation or malformation. The severity of the variation was also determined and assigned a score of 1 to 4; all malformations were assigned a score of 5.
Statistics:
Analyses of the log transformed pup body weights were conducted using a multilevel linear model. Pup sex and dam exposure group were included in the model and tested for significance. Log-transformed dam body weights were analysed by a multivariate repeated measures ANOVA. Viability data was analysed using a one-factor ANOVA.All post-hoc comparions were conducted using Dunnett's test. Skeletal, visceral and cephalic anomaly data were analysed by a log-linear model.
Indices:
Indices were not calculated.
Historical control data:
Historical control data were referred to when evaluating foetal anomalies.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Red material on the nose and face, salivation and scaly tail was seen in all treated rats including the controls.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Details on maternal toxic effects:
Maternal toxic effects:no effectsDetails on maternal toxic effects:There were no maternal mortalities. Red material on the nose and face, salivation and scaly tail was seen in all treated rats including the controls. No statistically significant differences in mean dam body or uterus weights, litter weights, or dam body weight gain were observed between PTA-exposed and control rats.
Dose descriptor:
NOAEC
Effect level:
10.4 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Effect level:
10.4 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effectsDetails on embryotoxic / teratogenic effects:There were no effects of treatment on pup viability.Similar numbers of resorptions were observed in all groups, with the control group having the highest number. The male to female ratio was approximately 1:1 in all groups. Observations noted during gross external examinations consisted of ablepharia, agnathia, exophthalmia, astomia and synotia in one control foetus and clubfoot in another control foetus. Two foetuses had short and/or filamentous tails; one foetus was in the 5 mg/m³ group (and also had an imperforate anus associated with the tail malformation), while the other foetus was in the 10 mg/m³ group. Another foetus in the 10 mg/m³ group had agnathia. Red marks on the foetuses were observed in all groups with similar incidences. The gross external anomalies observed were low in incidence, sporadic in nature, and within the historical control range for Charles River CD rats, therefore they were considered to be unrelated to PTA.No significant differences were detected in litter weights of the exposed rats compared to controls.The most prominent anomalies noted at wet visceral evaluation were hydroureter and/or hydronephrosis. These conditions were observed with similar incidences in all groups and were considered as normal variations of development. One foetus in the 10 mg/m³ group had fused kidneys with mispositioned ureters and ovaries. This foetus was also grossly malformed (i.e had a filamentous tail). Other anomalies observed were sporadic in nature and were not considered to be treatment related.172 foetuses were assigned to skeletal evaluation in the 10 mg/m³ group, however one foets was not read due to a processin artifact. Anomalies detected during skeletal examinations consisted of incomplete ossification of bones in the sternebrae, ribs, vertebral arches, pelvic girdle, digits and skull. The unossified skull bone was classified as a severe variation, but it was thought the observation may have been due to a processing artifact. Enlarged sutura and misshapen centrae and ribs were also observed. Additionally, rudimentary 14th rib or incomplete ossification of the ribs and centrae were observed. The incidence of the aforementioned anomalies was similar in the control and PTA-exposed groups. Several anomalies were observed in the ribs; independently these findings were not significant, but when all rib variations were collapsed together the 5 mg/m³ foetuses had a statistically significant increase in rib anomalies. Overall, most of the rib anomalies were considered to be normal variations, based on in-house historical control values and on the fact that no other signs of embryotoxicity were observed in any of the exposed groups. 42% of the foetuses with rib anomalies were in one litter.Four foetuses has anomalies which were classified as malformations: gnarled ribs in one foetus of the 5 mg/m³ group; misaligned sacral vertebrae/absent cauda centra in one 5 mg/m³ foetus; agnathia/general malformed skull was observed in one 10 mg/m³ foetus, while another foetus in the control group had a reduced jaw. The foetus with the reduced jae was a little-mate of the grossly malformed control foetus. All other anomalies detected were considered as slight variations, within normal limits, with similar incidences across all groups.One control foetus had mishappen nares, absent palate, convoluted retina, absent conjunctival sac and absent teeth. These observations correlated with the gross external description of this foetus. No other cephalic abnormalities were observed.
Dose descriptor:
NOAEC
Effect level:
10.4 mg/m³ air (analytical)
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Mean (± SD) analytical chamber concentrations for the 0, 1, 5 and 10 mg/m³ target concentrations were: 0, 0.99 ± 0.21, 5.30 ± 1.08 and 12.17 ± 2.08 mg/m³, respectively. The MMAD (mean of 3 samples ± SD) for the 1, 5 and 10 mg/m³ concentrations were: 4.16 ± 0.112; 4.87 ± 0.188 and 5.39 ± 0.200 microns, respectively. The percentage of respirable particles (≤ 10 um) for the 1, 5 and 10 mg/m³ concentrations was 91.1, 89.4 and 85%, respectively. The time-weighted average concentrations were 0, 0.9, 4.7, and 10.4 mg/m³ for the 0, 1, 5 and 10 mg/m³ groups respectively.

Conclusions:
No evidence of maternal or developmental toxicity was seen in an inhalation study in this study in rats exposed to terepthalic acid at levels of up to 10.4 mg/m³.
Executive summary:

In an effort to assess the teratogenic potential of purified terepthalic acid, the substance was administered as a particulate aerosol by inhalation at target concentrations of 0,1.0, 5.0, and 10.0 mg/m³ to four groups of 22 to 25 timed-pregnant primiparous Sprague-Dawley rats. The rats were exposed for 6 hours/day, on gestation Days 6 -15. The time-weighted average concentrations of the test material were 0, 0.9, 4.7, and 10.4 mg/m³ for the filtered air control, low, medium, and high exposure groups, respectively. No deaths occurred and no signs of maternal toxicity were observed during the study. No statistically significant differences in mean dam body or uterus weights, litter weights, dam body weight gain or pup viability were detected in the exposed rats compared to controls. External and soft tissue examinations failed to show any significant increase in the incidence of foetal malformations or abnormalities in the exposed litters compared to the controls. A statistically significant increase in the incidence of foetuses with rib anomalies was detected in the 5.0 mg/m³ group only when all rib anomalies were collapsed together. However, this increase was not considered a teratogenic event since the anomalies seen were common variations that were not elevated in a dose-related manner, were consistent with in-house historical control values, and no other signs of embryotoxicity were evident in the exposed groups. Therefore, exposure to 0.9, 4.7 or 10.4 mg/m³ terephthalic acid did not result in significant toxic or teratogenic effects in the dam or foetus.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
198 mg/kg bw/day
Study duration:
subacute
Species:
mouse
Quality of whole database:
High quality, GLP-compliant and guideline-compliant stuides are available for the read-across substance DEHT/DOPT in the rat and mouse.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
10 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
The studyy is reliable, but is limited by the relatively low exposure level used.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

A weight of evidence approach is taken to address the data requirements for developmental toxicity, using data for terephthalic acid (TPA) and the read-across substance DEHT/DOTP, which is shown to be hydrolysed to TPA following oral administration.

Inhalation developmental toxicity study with TPA

No evidence of developmental toxicity was seen in an inhalation study in rats exposed to TPA at levels of up to 10 mg/m3 (Ryan et al, 1990).

Oral developmental toxicity studies with DEHT/DOTP

A GLP- and Guideline-compliant study with the read-across substance DEHT in the rat showed no evidence of developmental toxicity following exposure to dietary concentrations of up to 10000 ppm DEHT on Gestation Days 0 -20. Maternal toxicity (reduced weight gain and increased liver weight) was seen in this study at the highest dietary concentration of 10000 ppm. An increased incidence of supernumerary rib seen at the highest concentration was considered to be treatment-related but not adverse. The highest dietary concentration of 10000 ppm DEHT is reported to be equivalent to 747 mg/kg bw/d and is calculated to be equivalent to 318 mg/kg bw/d terephthalic acid, based on the hydrolysis of DEHT to TPA.

A GLP- and Guideline-compliant study with the read-across substance DEHT in the mouse showed no evidence of developmental toxicity following exposure to dietary concentrations of up to 7000 ppm DEHT on Gestation Days 0 -18. Maternal toxicity (increased liver weight) was seen in this study at dietary concentrations of 3000 and 7000 ppm. The highest dietary concentration of 7000 ppm DEHT is reported to be equivalent to 1382 mg/kg bw/d and is calculated to be equivalent to 587 mg/kg bw/d terephthalic acid, based on the hydrolysis of DEHT to TPA.

Study of the effects of DEHT/DOTP on sexual differentiation

The effects of a number of phthalate esters on the sexual differentiation of male rats offspring (due to reported anti-androgenic properties) was assessed in a published study (Gray et al., 2000). In contrast to other substances investigated, the administration of DEHT at a dose level of 750 mg/kg bw/d to maternal female rats from Gestation Day 14 to Lactation Day 3 had no effect on sexual differentation, showing the absence of anti-androgenic activity for this substance.

Developmental toxicity studies: toxicokinetic considerations

It is also relevant that toxicokinetics studies (Tyl et al, 1982) indicate only limited placental transport of TPA following intravenous injection, due to the rapid maternal urinary excretion of the substance. Exposure of the developing embryo/foetus to TPA as a result of maternal exposure is therefore likely to be very low.


Justification for selection of Effect on developmental toxicity: via oral route:
A weight of evidence approach is taken to this endpoint, using read-across data from studies in two species (rat and mouse), neither of which reports any developmental toxicity at the highest dose levels tested. The rat study reports the lower NOAEL and is therefore selected. The NOAEL in the rat study (performed with DEHT) is corrected to show the equivalent TPA dose level based on the hydrolysis of DEHT to form TPA.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Only study available for this endpoint

Toxicity to reproduction: other studies

Additional information

Zhang et al. (2010) report adverse effects on the testes of male mice exposed to waste water (containing TPA and a number of other organic and inorganic pollutants) for 35 days. A slight (but statistically significant) reduction in the proportion of viable spermatogenic cells was seen in the treated group. Findings were accompanied by an increased number of abnormal sperm and testicular histopathology including expanded interstitial space, reduced Leydig cell size and reduced numbers of sperm. The results of this study indicate that the wastewater caused effects on the testes of male mice; however due to the complex nature of the material tested, the contribution of terephthalic acid to the observed effects is unknown. Comparing the results of this study to the wider reproductive toxicity dataset, it would seem to be extremely unlikely that the dose level of TPA achieved in this study (~3 mg/kg bw) is responsible for the effects observed. The study is not considered to be of direct relevance to the toxicological assessment of TPA due to the complex nature of the test material and is therefore disregarded.

Justification for classification or non-classification

A published 90 -day rat study (Cui et al, 1984) reports effects on sperm development (identified using electron microscopy) at the highest dose level of 5% in the diet and effects on sperm motility at lower dose levels. In contrast, it is notable that a reproductive screening study (Ledoux et al, 1982) reports no effects on reproductive parameters at dose levels of up to 5% in the diet. The Ledoux et al study notes marked bodyweight effects at the 5% dose level, with a NOAEL for bodyweight effects of 0.125%. Findings are consistent with the results of other 90 -day dietary toxicity studies, which note effects at comparable dose levels. In the 90 -day study of Vogin (1972), the 5% dose level was not tolerated and was subsequently reduced to 3%. In contrast, the paper of Cui et al does not note any bodyweight effects. A modern guideline-compliant 2 -generation study (Milburn et al, 2003) does not report any evidence of reproductive toxicity (including sperm parameters) in rats administered TPA at levels of up to 20000 ppm in the diet.

The findings of Cui et al (1984) were noted at a dietary concentration exceeding the limit concentration (1000 mg/kg bw/d) recommended for a reproductive toxicity study and at a concentration which, in a number of other dietary studies in the rat, has caused marked effects on bodyweight. Effects on sperm parameters were not noted in a guideline-compliant 2 -generation study and no effects on fertility or reproduction were seen in a one-generation and in a two-generation study.

No classification is therefore proposed for effects on fertility or reproduction. Findings in the Cui et al (2004) study were not reproducible and were seen at dose levels above which the production of an adverse effect is considered to be outside the criteria which lead to classification. No classification is proposed for developmental toxicity in the absence of any relevant findings in an inhalation study in the rat (Ryan et al, 1990).