Registration Dossier

Administrative data

Endpoint:
toxicity to reproduction: other studies
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The findings of this study are considered to be valid; however the nature of the test material (a complex mixture of substances including terephthalic acid) is such that the findings cannot be attributed to TPA or any individual component.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Assessment of the effects of manufacturing wastewater on the reproductive tract of male mice following repeated oral exposure.
GLP compliance:
no
Type of method:
in vivo

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
The test material (wastewater) was collected from a petrochemical wastewater treatment plant. The wastewater was characterised by GC/MS for organic compounds and by ICP for metal ions. Components are stated to be:

COD 9072 mg/L
BOD 4086 mg/L
pH 6.2
Total N 52 mg/L
Total P 9 mg/L
TSS 440 mg/L
Terephthalic acid 444 mg/L
p-Toluic acid 332 mg/L
Benzoic acid 227 mg/L
Phthalic acid 325 mg/L
p-Carbooxybenzaldehyde 18 mg/L
Ca 38 mg/L
Cu <0.002 mg/L
Mn 11 mg/L
Pb 0.44 mg/L
Zn 0.02 mg/L

Test animals

Species:
mouse
Strain:
other: KM
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Qinglong Hill Experimental Animal Centre, Nanjing, China
- Age at study initiation: no data
- Weight at study initiation: 18-22 g
- Fasting period before study: no data
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-55
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on exposure:
Male mice were administered wastewater or PBS (0.2 mL/day) on 35 consecutive days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test material (wastewater) was collected from a petrochemical wastewater treatment plant. The wastewater was characterised by GC/MS for organic compounds and by ICP for metal ions.
Duration of treatment / exposure:
35 days
Frequency of treatment:
Daily
Duration of test:
36 days
Doses / concentrations
Remarks:
Doses / Concentrations:
2 mL/day
Basis:
actual ingested
No. of animals per sex per dose:
12 males
Control animals:
yes
Details on study design:
Male mice were administered wastewater or PBS (0.2 mL/day) on 35 consecutive days; mice were sacrificed on Day 36.
Statistics:
Results were analysed using the independent samples t-test (p<0.05)

Results and discussion

Observed effects

No deaths occurred. Final bodyweights of treated mice were 18% higher than controls; the difference was not statistically significant. Relative liver and testes weights were significantly lower than controls (12.4% and 10,6%, respectively); however these values may be seondary to bodyweight effects. Flow cytometry showed significant reductions in the proportions of 2C and SC cells and a reduced 1C:4C ratio. 1C:2C, 4C:2C and 4C:SC cell ratios were significantly increased in the treated group. Samples from the treated group were shown to have a significantly higher proportion of abnormal sperm including folded tail, absent head and folded neck. The proportion of viable spermatogenic cells was slightly (but significantly) reduced in the treated group. Testicular histopathology of treated mice revealed an expanded interstitial space, reduced Leydig cell size and reduced numbers of sperm.

Any other information on results incl. tables

 

Controls

Wastewater treated

Initial bodyweight (g)

26.46

26.46

Final bodyweight (g)

29.09

34.42

Relative liver weight

5.52%

4.83%**

Relative testes weight

0.74%

0.66%*

Spermatogenic cells

1C

66.9

68.9

2C

20.7

19.5*

SC

7.5

2.2**

4C

3.6

7.7**

1C:4C

19.3

9.0**

1C:2C

3.2

3.5*

4C:2C

0.17

0.39**

4C:SC

0.48

3.5**

Abnormal sperm

3.09%

6.22%*

Viable spermatogenic cells

93.1%

90.4%*

*significantly different to controsl (p<0.05) **p<0.01

Applicant's summary and conclusion

Conclusions:
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.
Executive summary:

Groups of 10 male KM mice were administered 0.2 mL petrochemical plant wastewater containing terephthalic acid (444 mg/L) and numerous other components. Based on the TPA content of the test material and the average bodyweight of the treated mice, TPA intake is calculated to be approximately 3 mg/kg bw/d. Bodyweights were recorded on Day 1 and Day 35. Weights of the heart, liver, lungs, spleen, kidney and testes were recorded and relative organ weights calculated. Spermatogenic cells were isolated from the testes, stained with propdidum iodide and analysed using flow cytometry. The relative proportions of haploid, diploid, S-phase and tetraploid cells were calculated based on staining intensity. Cell viability was assessed based on the measurement of intracellular lipase activity and plasma membrane integrity. Epididymal sperm were assessed morpholoogically. The testes were assessed histopathologically following fixation in Bouin's. No deaths occurred. Final bodyweights of treated mice were 18% higher than controls; the difference was not statistically significant. Relative liver and testes weights were significantly lower than controls (12.4% and 10,6%, respectively); however these values may be seondary to bodyweight effects. Flow cytometry showed reductions in the proportions of 2C and SC cells and a reduced 1C:4C ratio. 1C:2C, 4C:2C and 4C:SC cell ratios were significantly increased in the treated group. Samples from the treated group were shown to have a significantly higher proportion of abnormal sperm including folded tail, absent head and folded neck. The proportion of viable spermatogenic cells was slightly (but significantly) reduced in the treated group. Testicular histopathology of treated mice revealed an 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 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.